Method of detecting cancer based on glycan biomarkers

ABSTRACT

The present invention provides a method for labeling or detecting a protein with certain glycosyl groups. The methods are particularly useful for detecting cancer cells comprising the detected glycosyl groups. The present invention further provides labeling agents and detection agents, labeled proteins and mixtures, and kits and arrays thereof.

CROSS-REFERENCE

This application claims the benefit under 35 USC §119(e) of U.S.Provisional Application No. 61/748,895, filed on Jan. 4, 2013, whichapplication is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R01 GM084724awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 3, 2014, isnamed 38075-720.201_SL.txt and is 15,023 bytes in size.

BACKGROUND OF THE INVENTION

The disaccharide motif fucose-α(1-2)-galactose (Fucα(1-2)Gal) isinvolved in many important physiological processes, such as learning andmemory, inflammation, asthma, and tumorigenesis. However, the size andstructural complexity of Fucα(1-2)Gal-containing glycans has posed asignificant challenge to their detection and study.

Defects in glycosylation are a hallmark of many human diseases,including autoimmune disorders, neurodegenerative diseases, and cancer.(Delves, P. J. Autoimmunity 1998, 27, 239; Rexach, J. E.; Clark, P. M.;Hsieh-Wilson, L. C. Nat. Chem. Biol. 2008, 4, 97; Kim, Y.; Varki, A.Glycoconjugate J. 1997, 14, 569.) Fucα(1-2)Gal is found on thenon-reducing terminus of a large family of important glycans, includingblood group H1 and H2, Globo H, Fuc-GM1, Lewis B, and Lewis Y.

SUMMARY OF THE INVENTION

The invention provides a method for labeling a glycan having a glycosylgroup comprising a fucose linked to a galactose. In some embodiments,the method comprises reacting the glycan with a labeling agent in thepresence of a glycosyltransferase to form a labeled glycan, wherein thelabeling agent comprises a transferable glycosyl group recognized by thetransferase and a reactive group capable of reacting with a detectionagent, and wherein the glycosyltransferase is specific for the glycosylgroup. In some embodiments, the method further comprises reacting thelabeled glycan with a detection agent comprising a coupling moiety tocovalently couple the detection agent to the labeling agent on theglycan. In some embodiments, the method further comprises detecting thedetection agent covalently bound to the protein via the reactive groupthereby detecting the presence of the protein. In some embodiments, heglycosyltransferase is specific for a fucose-α(1-2)-galactose group. Insome embodiments, the glycan is a glycoprotein or glycolipid. In someembodiments, the glycosyltransferase is a bacteria homologue of thehuman blood group A antigen glycosyltransferase (BgtA) or a variant orfragment thereof. In some embodiments, the glycosyltransferase is aglycosyl transferase having 95% sequence identity to SEQ ID NO:1 or SEQID NO:2. In some embodiments, the glycosyl group comprises afucose-α(1-2)-galactose group. In some embodiments, the detection agentrecruits another agent selected from the group consisting of a secondarylabeling agent, an enzyme, and a secondary detection agent. In someembodiments, the detection agent is biotin or biotin derivative. In someembodiments, the reactive group is selected from the group consisting ofcarbonyl group, azide group, nitril oxide group, diazoalkane group,alkyne group, and olefin group. In some embodiments, the detection agentcomprises a coupling moiety selected from the group consisting of —C═C—(alkene), —C≡C— (alkyne), —NR¹—NH₂ (hydrazide), —NR¹ (C═O)NR²NH₂(semicarbazide), —NR¹ (C═S)NR²NH₂ (thiosemicarbazide), —(C═O) NR¹NH₂(carbonylhydrazide), —(C═S) NR¹NH₂ (thiocarbonylhydrazide), —(SO₂)NR¹NH₂ (sulfonylhydrazide), —NR¹NR² (C═O)NR³NH₂ (carbazide), —NR¹NR²(C═S)NR³NH₂ (thiocarbazide), and —O—NH₂ (aminooxy), wherein each R¹, R²,and R³ is independently H or alkyl having 1-6 carbons. In someembodiments, the detecting step is achieved by a means selected from thegroup consisting of radioactively, chemiluminescent, fluorescent, massspectrometric, spin-labeling, and affinity labeling. In someembodiments, the labeling agent has the formula I:

wherein R is a substituent selected from the group consisting ofstraight chain or branched C₁-C₁₂ carbon chain bearing a carbonyl group,azide group, straight chain or branched C₁-C₁₂ carbon chain bearing anazide group, straight chain or branched C₁-C₁₂ carbon chain bearing analkyne, straight chain or branched C₁-C₁₂ carbon chain bearing analkene, and —NHC(O)CH₂N₃. In some embodiments, the labeling agent hasthe formula II:

or formula III:

Also provided herein is a labeled glycan obtained by the method of thepresent invention.

Also provided herein is a labeled glycan comprising 1) a first glycosylgroup comprising a fucose linked to a galactose; and 2) a secondglycosyl group covalently linked to the first glycosyl group, whereinthe second glycosyl group comprises a reactive group. In someembodiments, the glycan is attached to a glycoprotein or glycolipid. Insome embodiments, the second glycosyl group is covalently linked to thefirst glycosyl group via the galactose on the first glycosyl group. Insome embodiments, the second glycosyl group is covalently linked to thefirst glycosyl group at C-3 position of the galactose on the firstglycosyl group. In some embodiments, the glycan further comprises adetection agent covalently linked to the second glycosyl group, whereinthe detection agent is covalently linked to the second glycosyl groupvia a reaction between a coupling moiety on the detection agent and thereactive group. In some embodiments, the first glycosyl group is afucose-α(1-2)-galactose. In some embodiments, the glycan has the formulaof

wherein R is a substitution comprising the reactive group.

In another aspect, the present invention provides a reaction mixture. Insome embodiments, the reaction mixture comprises (1) a glycan having aglycosyl group comprising a fucose linked to a galactose, and (2) alabeling agent comprising a transferable glycosyl group recognized by atransferase capable of transfer the group to the glycoprotein, and areactive group. In some embodiments, the glycan is attached to aglycoprotein or glycolipid. In some embodiments, the reaction mixturefurther comprises a glycosyltransferase specific for the glycosyl groupon the glycoprotein. In some embodiments, the reaction mixture furthercomprises a detection agent, wherein the detection agent comprises acoupling moiety that is capable of reacting with the reactive group onthe labeling agent to form a covalent bond. In some embodiments, theglycosyltransferase is specific for a fucose-α(1-2)-galactose group. Insome embodiments, the glycosyl transferase is a bacteria homologue ofthe human blood group A antigen glycosyltransferase (BgtA) or a variantor fragment thereof. In some embodiments, the glycosyltransferase is ahuman blood group A antigen glycosyltransferase or a variant or fragmentthereof. In some embodiments, the glycosyl group comprises afucose-α(1-2)-galactose group. In some embodiments, the labeling agenthas the formula I:

wherein R is a substituent selected from the group consisting ofstraight chain or branched C₁-C₁₂ carbon chain bearing a carbonyl group,azide group, straight chain or branched C₁-C₁₂ carbon chain bearing anazide group, straight chain or branched C₁-C₁₂ carbon chain bearing analkyne, straight chain or branched C₁-C₁₂ carbon chain bearing analkene. In some embodiments, the labeling agent has the formula II:

In some embodiments, the labeling agent has the formula III:

In some embodiments, the reaction mixture is placed on a solid support.

In another aspect, the present invention provides a kit for labeling aglycan with a glycosyl group comprising a fucose linked to a galactose.In some embodiments, the kit comprises: (a) a glycosyltransferase,wherein the glycosyltransferase is specific for the glycosyl groupcomprising a fucose linked to a galactose and is capable of catalyzingthe transfer of a transferable glycosyl group on a labeling agent to theglycosyl group on the glycan; and (b) instructions instructing a user toperform the labeling using component (a). In some embodiments, theglycan is attached to a glycoprotein or glycolipid. In some embodiments,the kit further comprises a labeling agent comprising a transferableglycosyl group recognized by the transferase, and a reactive group. Insome embodiments, the kit further comprises a detection agent comprisinga coupling moiety capable of reacting with the reactive group on thelabeling agent to form a covalent bond. In some embodiments, theglycosyl transferase is specific for a fucose-α(1-2)-galactose group. Insome embodiments, the glycosyltransferase is a bacteria homologue of thehuman blood group A antigen glycosyltransferase (BgtA) or a variant orfragment thereof. In some embodiments, the glycosyltransferase is ahuman blood group A antigen glycosyltransferase or a variant or fragmentthereof. In some embodiments, the glycosyl group of the glycoproteincomprises a fucose-α(1-2)-galactose group. In some embodiments, thedetection agent is selected from the group consisting of fluorescentreagent, enzymatic reagent capable of converting substratescalorimetrically or fluorometrically, fluorescent and luminescent probe,metal-binding probe, protein-binding probe, probe for antibody-basedbinding, radioactive probe, photocaged probe, spin-label orspectroscopic probe, heavy-atom containing probe, polymer containingprobe, probe for protein cross-linking, and probe for binding toparticles or surfaces that contain complementary functionality. In someembodiments, the detection agent is biotin or biotin derivative. In someembodiments, the reactive group is selected from the group consisting ofcarbonyl group, azide group, nitril oxide group, diazoalkane group,alkyne group, and olefin group. In some embodiments, the detection agentcomprises a coupling moiety selected from the group consisting of —C═C—(alkene), —C≡C— (alkyne), —NR¹—NH₂ (hydrazide), —NR¹ (C═O)NR²NH₂(semicarbazide), —NR¹ (C═S)NR²NH₂ (thiosemicarbazide), —(C═O) NR¹NH₂(carbonylhydrazide), —(C═S) NR¹NH₂ (thiocarbonylhydrazide), —(SO₂)NR¹NH₂ (sulfonylhydrazide), —NR¹NR² (C═O)NR³NH₂ (carbazide), —NR¹NR²(C═S)NR³NH₂ (thiocarbazide), and —O—NH₂ (aminooxy), wherein each R¹, R²,and R³ is independently H or alkyl having 1-6 carbons. In someembodiments, the labeling agent has the formula I:

wherein R is a substituent selected from the group consisting ofstraight chain or branched C₁-C₁₂ carbon chain bearing a carbonyl group,azide group, straight chain or branched C₁-C₁₂ carbon chain bearing anazide group, straight chain or branched C₁-C₁₂ carbon chain bearing analkyne, straight chain or branched C₁-C₁₂ carbon chain bearing analkene. In some embodiments, the labeling agent has the formula II:

In some embodiments, the labeling agent has the formula III:

In some embodiments, the kit further comprises a separation device forpurifying a labeled glycan.

In another aspect, the present invention provides a method ofidentifying a glycan comprising a fucose linked to a galactose. In someembodiments, the method comprises 1) providing one or more homogenouspopulation of glycans on a solid support; 2) contacting aglycosyltransferase with the glycans in the presence of a labeling agentcomprising a transferable glycosyl group, wherein the transferableglycosyl group comprises a reactive group, wherein theglycosyltransferase is specific for the glycosyl group comprising afucose linked to a galactose and catalyzes the transfer of thetransferable glycosyl group to the glycosyl group comprising a fucoselinked to a galactose; 3) contacting the glycans with a detection agent,the reactive group on the transferred glycosyl group reacts with acoupling moiety on the detection agent to form a covalent bond; and 4)identifying a glycan having the covalently bound detection agent via thereactive group on the transferred glycosyl group as the glycancomprising a fucose linked to a galactose. In some embodiments, theglycans are attached to the solid support in the form of an arraycomprising one or more addressable locations.

In another aspect, the present invention provides a method of detectingcancer cells expressing a glycoprotein comprising a glycosyl groupcomprising a fucose linked to a galactose. In some embodiments, themethod comprises the steps of: 1) contacting the cell with aglycosyltransferase and a labeling agent, the labeling agent comprises atransferable glycosyl group that is transferable by the transferase tothe glycoprotein, wherein the transferable glycosyl group comprises areactive group capable of reacting with a coupling moiety of a detectionagent; 2) contacting the cell with a detection agent, wherein thereactive group on a transferred glycosyl group reacts with a couplingmoiety on the detection agent to effect covalent coupling of thedetection agent to the labeling agent; 3) detecting the amount of thedetection agent covalently bound to the cell via the reactive group onthe transferred labeling agent; and 4) comparing the amount of thedetection agent covalently bound to the cell to the amount of thedetection agent covalently bound in a non-cancerous control. An increasein the amount of the detection agent covalently bound to the cell ascompared to the amount of the detection agent covalently bound in anon-cancerous control indicates a presence of cancer cells having theglycosyl group comprising a fucose linked to a galactose. In someembodiments, the detection agent recruits another agent selected fromthe group consisting of a labeling agent, an enzyme, and a secondarydetection agent. In some embodiments, the detection agent is biotin orbiotin derivative. In some embodiments, the biotin or biotin derivativerecruits a secondary detection agent selected from the group consistingof fluorescent reagent, enzymatic reagent capable of convertingsubstrates colorimetrically or fluorometrically, fluorescent andluminescent probe, metal binding probe, protein-binding probe, probe forantibody-based binding, radioactive probe, photocaged probe, spin-labelor spectroscopic probe, heavy-atom containing probe, polymer containingprobe, probe for protein cross-linking, and probe for binding toparticles or surfaces that contain complementary functionality. In someembodiments, the reactive group is selected from the group consisting ofcarbonyl group, azide group, nitril oxide group, diazoalkane group,alkyne group, and olefin group. In some embodiments, the reactive groupis a carbonyl group. In some embodiments, the reactive group is an azidegroup. In some embodiments, the detection agent comprises a couplingmoiety selected from the group consisting of —C═C— (alkene), —C≡C—(alkyne), —NR¹—NH₂ (hydrazide), —NR¹ (C═O)NR²NH₂ (semicarbazide),—NR¹(C═S)NR²NH₂ (thiosemicarbazide), —(C═O) NR¹NH₂ (carbonylhydrazide),—(C═S) NR¹NH₂ (thiocarbonylhydrazide), —(SO₂) NR¹NH₂(sulfonylhydrazide), —NR¹NR² (C═O)NR³NH₂ (carbazide), —NR¹NR²(C═S)NR³NH₂ (thiocarbazide), and —O—NH₂ (aminooxy), wherein each R¹, R²,and R³ is independently H or alkyl having 1-6 carbons. In someembodiments, the detection agent comprises a coupling moiety selectedfrom the group consisting of —C═C— (alkene), —C≡C— (alkyne), hydrazide,aminooxy, semicarbazide, carbohydrazide, and sulfonyihydrazide. In someembodiments, the cancer is selected from the group consisting of breastcancer, lung cancer, prostate cancer, colon cancer, colorectal cancer,cervical cancer, and pancreatic cancer.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1. (A) Chemoenzymatic strategy for the detection of Fucα(1-2)Galglycans. (B) Labeling of substrate 1. LC-MS traces monitoring thereaction progress at time 0 (top), 12 h after the addition of BgtA and 2(middle), and 3 h after the addition of ADIBO-biotin 6 (bottom). See SIfor details.

FIG. 2. (A) Time course analysis using glycan microarrays.Representative structures from the top 26 glycans with the highestrelative fluorescence intensity after 0.5 h are plotted, all of whichrepresent terminal Fucα(1-2)Gal structures. UDP-GalNAz was omitted fromsome of the reactions as a control (12 h, -UDP-GalNAz). (B)Chemoenzymatic detection of endogenous Fucα(1-2)Gal glycoproteins fromneuronal lysates. (C) Chemoenzymatic detection of Flag-tagged synapsin Iexpressed in HeLa cells. See SI for experimental details.

FIG. 3. (A) Fluorescence detection of Fucα(1,2)Gal glycans (green) inHeLa cells shows excellent co-localization (yellow) with Flag-taggedsynapsin I (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole(DAPI; blue). (B) Fluorescence detection of Fucα(1,2)Gal glycans (green)on live MCF-7 cells. Nuclei were stained with Hoechst 342 (blue).

FIG. 4. Flow cytometry analysis of the relative expression levels ofFucα(1,2)Gal glycans across various cancer cell lines, with comparisonto non-cancerous PrEC cells. Cells were untreated (red) orchemoenzymatically labeled in the presence (blue) or absence (green) ofBgtA. Quantification of the mean fluorescence intensity (MFI) relativeto cells labeled in the absence of BgtA is shown on the right. Errorbars represent data from duplicate (MCF-7, MDA-mb-231, H1299) ortriplicate (LnCAP, PrEC) experiments.

FIG. 5. Synthesis of Fucα(1-2)Gal substrate 1.

FIG. 6. (A) Chemoenzymatic labeling of substrate 1 with UDP-ketoGal 3.LC-MS analysis of the reaction progress at time 0 (top), 12 h after theaddition of BgtA and 3 (middle), and 24 h after the addition ofaminooxy-biotin derivative 7 (bottom). See Materials and Methods fordetails. (B) Compounds used for chemoenzymatic labeling of glycans.

FIG. 7. LC-MS/MS analysis of 1, 4, and 8 during the chemoenzymaticlabeling reaction. (A) Compound 1 at time 0. (B) Compound 4, generated12 h after the addition of BgtA and UDP-GalNAz 2. (C) Biotinylatedglycan 8, generated 3 h after reaction with ADIBO-biotin 6. The MSspectrum for each compound is shown on top; the MS/MS spectrum for themost abundant ion is shown on the bottom. The m/z of peaks found in eachMS/MS analysis are shown as either b and y or c and z ions. Thecorresponding fragmentation products and probable cleavage sites aredenoted in the respective structures. 1 and 4 were detected in positivescanning mode; 8 was detected in negative scanning mode.

FIG. 8. Kinetic comparison of the BgtA-catalyzed reaction of 1 withUDP-GalNAc (black) and UDP-GalNAz (blue). Reactions were performed induplicate using 100 μM of acceptor 1 and varying concentrations of thedonor. Initial rates as a function of substrate concentration wereplotted and revealed apparent k_(cat)/K_(m) values of 5.7 nM⁻¹ min⁻¹ and40.4 nM⁻¹ min⁻¹, respectively, and apparent K_(m) values of 127±36 μMand 168±55 μM, respectively. The apparent V_(max) value for UDP-GalNAc(0.100±0.010 nmol·min⁻¹) is approximately 5-fold higher than that ofUDP-GalNAz (0.020±0.002 nmol·min⁻¹).

FIG. 9. Additional Fucα(1-2)Gal structures on the microarray and theirability to be labeled by BgtA (see also FIG. 2A). Relative fluorescenceintensities are plotted as a function of time and represent the mean of4 values. Error bars represent the standard deviation of the mean.Glycans were considered labeled if they showed a time-dependent increasein fluorescence labeling and their signal at 12 h after subtraction ofthe background in the absence of UDP-GalNAz (12 h, -UDP-GalNAz)was >1000 relative fluorescence units. The red asterisks indicatestructures that were not considered to be labeled. W indicates very weaklabeling.

FIG. 10. The chemoenzymatic approach labels a variety of linear (A) andbranched (B) Fucα (1-2)Gal structures. (A) The third sugar toward thereducing end of the glycan does not significantly affect the labelingreaction (eg. 62, 66, 74, and 78). Acceptor substrate structures thathave a GlcNAc, instead of Gal, and change the linkage from α(1-2) toα(1-3), α(1-4), or β(1-3) are not modified by BgtA (eg 80, 81, and 82).Representative structures from the microarray are shown for comparison.Relative fluorescence intensities are plotted as a function of time toshow how subtle changes in the structure affect the kinetics oflabeling. Error bars represent the standard deviation of the mean of 4values after removing the high and low values from n=6 replicates ofeach glycan printed on the array.

FIG. 11. The chemoenzymatic approach labels a variety of Fucα(1-2)Galstructures. (A) Branching at the third position GlcNAc via α(1-3) orα(1-4) fucosylation severely hindered the labeling efficiency. (B) Weaklabeling of Galβ(1-4)GlcNAc structures was also observed. This labelingwas accompanied by high background, as indicated by the 12 h time pointin the absence of UDP-GalNAz. No labeling of these structures wasobserved in solution, suggesting that the labeling was likely due tonon-covalent interactions with the microarray. Representative structuresare shown for comparison. Relative fluorescence intensities are plottedas a function of time to show how changes in glycan structure affect thelabeling reaction. Error bars represent the standard deviation of themean of 4 values after removing the high and low values from n=6replicates of each glycan printed on the array.

FIG. 12. In-gel fluorescence detection of Fucα(1-2)Gal glycoproteinsfrom neuronal cell lysates. Low background fluorescence was observed inthe absence of BgtA, UDP-GalNAz 2, or alkyne-TAMRA 10. The band at ˜35kDa is BgtA, which appears to label itself.

FIG. 13. Comparison of UEAI lectin affinity chromatography to thechemoenzymatic strategy. Glycosylated synapsin I from olfactory bulblysate (500 μg) was subjected to lectin affinity chromatography orchemoenzymatic labeling followed by streptavidin capture. Westernblotting for synapsin I indicated that UEAI failed to capture and detectglycosylated synapsin I, whereas the chemoenzymatic strategy allowed forready detection.

FIG. 14. Low fluorescence labeling of endogenous Fucα(1-2)Galglycoproteins in HeLa cells. Cells were mock-transfected with an emptyFLAG-vector and chemoenzymatically labeled with UDP-GalNAz and BgtA,followed by Alexa-Fluor (AF) 488 alkyne. Weak labeling of theglycoproteins (green) was observed in cells not transfected withsynapsin I, suggesting low expression levels of endogenous Fucα(1-2)Galglycoproteins. Nuclei were stained with 4′,6-diamidino-2-phenylindole(DAPI; blue).

DEFINITIONS

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

A “subject,” “individual” or “patient” is used interchangeably herein,which refers to a vertebrate, in some embodiments a mammal, in someembodiments a human. Mammals include, but are not limited to mice, rats,dogs, pigs, monkey (simians) humans, farm animals, sport animals, andpets. Tissues, cells and their progeny of a biological entity obtainedin vivo or cultured in vitro are also encompassed.

As used herein, “labeling agent” is an agent that can react with aglycosyl group comprising a fucose linked to a galactose (e.g., afucose-α(1-2)-galactose group). In some embodiments, a labeling agentcan further comprise a reactive group for further elaboration ordetection.

As used herein, “reactive group” is a functional group. In someembodiments, the reactive group can be one of a number of groups as setforth below that can react in a selective manner with a detection agentvia a coupling moiety in the presence of various biomolecules.Alternatively, the reactive group can itself comprise a detection agent.Such detection agent can be a radioactive atom, as described below.

As used herein and described below, “coupling moiety” is a functionalmoiety that undergoes a chemical reaction with the reactive group. Acoupling moiety can be contained on a detection agent to react with thereactive group.

As used herein, “detection agent” is an agent that has a property thatcan be observed spectroscopically or visually. Methods for production ofdetectably labeled proteins using detection agents are well known in theart. Detectable labels include, but are not limited to, radioisotopes,fluorophores, paramagnetic labels, antibodies, enzymes (e.g.,horseradish peroxidase), or other moieties or compounds which eitheremit a detectable signal (e.g., radioactivity, fluorescence, color) oremit a detectable signal after exposure of the detection agent to itssubstrate.

A “variant” is a protein with sequence homology to the nativebiologically active protein that retains at least a portion of thetherapeutic and/or biological activity of the biologically activeprotein. For example, a variant protein may share at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identitycompared with the reference biologically active protein. As used herein,the term “biologically active protein” includes proteins modifieddeliberately, as for example, by site directed mutagenesis, insertions,or accidentally through mutations. A “variant” includes a “fragment”,which is a truncated form of a native or non-native biologically activeprotein that retains at least a portion of the therapeutic and/orbiological activity.

The term “sequence variant” means polypeptides that have been modifiedcompared to their native or original sequence by one or more amino acidinsertions, deletions, or substitutions. Insertions may be located ateither or both termini of the protein, and/or may be positioned withininternal regions of the amino acid sequence. In deletion variants, oneor more amino acid residues in a polypeptide as described herein areremoved. In substitution variants, one or more amino acid residues of apolypeptide are removed and replaced with alternative residues. In oneaspect, the substitutions are conservative in nature and conservativesubstitutions of this type are well known in the art.

“Percent (%) sequence identity,” with respect to the polypeptidesequences identified herein, is defined as the percentage of amino acidresidues in a query sequence that are identical with the amino acidresidues of a second, reference polypeptide sequence or a portionthereof, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Percentidentity may be measured over the length of an entire definedpolypeptide sequence, or may be measured over a shorter length, forexample, over the length of a fragment taken from a larger, definedpolypeptide sequence, for instance, a fragment of at least 15, at least20, at least 30, at least 40, at least 50, at least 70 or at least 150contiguous residues. Such lengths are exemplary only, and it isunderstood that any fragment length supported by the sequences shownherein, in the tables, figures or Sequence Listing, may be used todescribe a length over which percentage identity may be measured.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for labeling(e.g., detecting) a protein having a glycosyl group comprising a fucoselinked to a galactose. The invention also provides a labeled proteinobtained from contacting the protein with a labeling agent andoptionally a detection agent. Typically, the labeling agent comprises areactive group and a transferable glycosyl group. Where desired, thereactive group is located on the transferable glycosyl group. Thereactive group on the transferred glycosyl group is capable of reactingwith a coupling moiety on the detection agent to form a covalent bond.An exemplary labeled protein comprises a first glycosyl group comprisinga fucose linked to a galactose and a second glycosyl group covalentlylinked to the first glycosyl group. The present invention furtherprovides methods, compositions, kits, and arrays for detecting certaindisease states, such as cancer.

Methods of Labeling or Detecting a Glycan

The present invention provides methods for labeling (e.g., detecting) aglycan (e.g., a glycoprotein), particularly glycan having a glycosylgroup comprising a fucose linked to a galactose. In some embodiments,the methods involve reacting the glycan with a labeling agent in thepresence of a glycosyltransferase to form a labeled glycan. The labelingagent comprises a transferable glycosyl group recognized by thetransferase, and further comprises a reactive group capable of reactingwith a detection agent. The glycosyltransferase is specific for aglycosyl group comprising a fucose linked to a galactose and cancatalyze the transfer of the transferable glycosyl group on the labelingagent to the glycosyl group comprising a fucose linked to a galactose ona glycan (e.g., a glycoprotein). A modified glycan results from reactionof the labeling agent with the glycosyl group comprising a fucose linkedto a galactose on the protein. In some embodiments, the glycosyl groupis a fucose-α(1-2)-galactose group. In some embodiments, theglycosyltransferase is specific for a fucose-α(1-2)-galactose group.Exemplary glycosyltransferases include a human blood group A antigenglycosyltransferase or a variant or fragment thereof, e.g., a bacteriahomologue of the human blood group A antigen glycosyltransferase (BgtA)or a variant or fragment thereof.

The labeling agent can further comprise a reactive group. The reactivegroup on the labeling agent can react with a detection agent via areaction between the reactive group and a coupling moiety on thedetection agent. Where desired, the reactive group does not reactsubstantially with a protein or other components of a biologicalmixture. In some embodiments, the detection agent is covalently coupledto the labeling agent after the labeling agent is conjugated to theglycan in the presence of a glycosyltransferase to form a labeledglycan. The reactive group on the labeling agent can be used to furtherreact the modified protein with a detection agent via a reaction betweenthe reactive group and a coupling moiety on the detection agent. In someembodiments, the detection agent is first covalently coupled to thelabeling agent. The resulting compound is then reacted with the glycanto form a labeled glycan.

The detection agent can be detectable through various detection means,such as, but not limited to, radioactively, chemiluminescence,fluorescence, mass spectrometry, spin labeling, affinity labeling, orthe like. The detection agent can be, for example, a radiolabeledcompound or a fluorescent compound. The detection agent also can bedetectable indirectly, for example, by recruitment of one or moreadditional factors.

In some embodiments, the glycan is attached to a glycoprotein orglycolipid. Glycoproteins comprise proteins covalently linked tocarbohydrate. The predominant sugars found in glycoproteins are glucose,galactose, mannose, fucose, GalNAc, GlcNAc and NANA. Carbohydrates canbe linked to the protein component through O-glycosidic, N-glycosidic,or C-glycosidic bonds. In some embodiments, the methods described hereinare useful for detecting glycosylated proteins. In some embodiments,certain post-translational modifications will append a glycosyl group.In some embodiments, the glycosyl group is a glycosyl group comprising afucose linked to a galactose. In some embodiments, proteins having aglycosyl group comprising a fucose linked to a galactose are detected.In some embodiments, proteins having a fucose-α(1-2)-galactose group aredetected. Changes in fucose-α(1-2)-galactose levels have been associatedwith disease states such as cancer.

In some embodiments, a labeling agent is an agent that can react with atarget glycosyl group of a glycan (e.g., a glycoprotein) while furthercomprising a reactive group for further reaction. A glycosyltransferasespecific for the target glycosyl group can be used to transfer atransferable glycosyl group on the labeling agent to the target glycosylgroup on the glycan of interest. The glycosyltransferase can be anaturally occurring glycosyltransferase, a mutant glycosyltransferase,or an evolved glycosyltransferase that is specific for the targetglycosyl group. In some embodiments, the glycosyltransferase is specificfor a glycosyl group comprising a fucose linked to a galactose. In someembodiments, the glycosyltransferase is specific for afucose-α(1-2)-galactose group.

Exemplary glycosyltransferases include, but are not limited to, a humanblood group A antigen glycosyltransferase or a variant or fragmentthereof. In some embodiments, glycosyltransferases include a bacteriahomologue of the human blood group A antigen glycosyltransferase or avariant or fragment thereof, e.g., a Helicobacter mustelae homologue ofthe human blood group A antigen glycosyltransferase or a variant orfragment thereof (Yi, W.; Shen, J.; Zhou, G.; Li, J.; Wang, P. G. J. Am.Chem. Soc. 2008, 130, 14420). In some embodiments, glycosyltransferasesare engineered to accept unnatural substrates. For example, in someembodiments, glycosyltransferases are engineered to tolerate unnaturalsubstrates containing substitutions at one or more positions on thesugar ring (e.g., the C-2 position).

In some embodiment, the glycosyltransferase is a variant (e.g., asequence variant) or fragment of a glycosyl transferase having SEQ IDNO:1, 2, 3, 4, or 5. In some embodiments, the glycosyltransferase is aglycosyltransferase having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% amino acid sequence identity to SEQ ID NO:1, 2, 3, 4, or5. In some embodiment, the glycosyltransferase is SEQ ID NO:1, 2, 3, 4,or 5.

The reactive group can be one of a number of groups that can react in aselective manner with the coupling moiety of a detection agent in thepresence of various biomolecules, and particularly in an aqueoussolution. Alternatively, the reactive group can itself comprise adetection agent. In some embodiments, the reactive group comprises aradioactive substance. A reactive group is contained on a labelingagent, e.g., on a transferable glycol group of the labeling agent.

In some embodiments, the reactive group is a carbonyl group reactivegroup. The carbonyl group participates in a large number of reactionsfrom addition and decarboxylation reactions to aldol condensations.Moreover, the unique reactivity of the carbonyl group allows it to beselectively modified with hydrazide and aminooxy derivatives in thepresence of the other amino acid side chains. See, e.g., Cornish, V. W.,Hahn, K. M. & Schultz, P. G. (1996) J. Am. Chem. Soc. 118:8150-8151;Geoghegan, K. F. & Stroh, J. G. (1992) Bioconjug. Chem. 3:138-146; and,Mahal, L. K., Yarema, K. J. & Bertozzi, C. R. (1997) Science276:1125-1128. This functional group is generally absent from proteinsand thus can serve as a reactive group for subsequent proteinmodification.

For reaction with the carbonyl group reactive group, a coupling moietycan be —NR¹—NH₂ (hydrazide), —NR¹(C═O)NR²NH₂ (semicarbazide),—NR¹(C═S)NR²NH₂ (thiosemicarbazide), —(C═O)NR¹NH₂ (carbonylhydrazide),—(C═S)NR¹NH₂ (thiocarbonylhydrazide), —(SO₂)NR¹NH₂ (sulfonylhydrazide),—NR¹NR² (C═O)NR³NH₂ (carbazide), —NR¹NR²(C═S)NR³NH₂ (thiocarbazide),—O—NH₂ (aminooxy), and/or the like, where each R¹, R², and R³ isindependently H, or alkyl having 1-6 carbons, in some embodiments H. Insome embodiments, the coupling moiety is a —C═C— (alkene), —C≡C—(alkyne), hydrazide, aninooxy, semicarbazide, carbohydrazide, asulfonylhydrazide, or the like.

The product of the reaction between the reactive group and the couplingmoiety typically incorporates the atoms originally present in thecoupling moiety. Typical linkages obtained by reacting the aldehyde orketone reactive groups with certain coupling moieties include but arenot limited to reaction products such as an oxime, a hydrazone, areduced hydrazone, a carbohydrazone, a thiocarbohydrazone, asulfonylhydrazone, a semicarbazone, a thiosemicarbazone, or similarfunctionality, depending on the nucleophilic moiety of the couplingmoiety and the aldehyde or ketone reactive group. Linkages withcarboxylic acids are also possible and result in carbohydrazides orhydroxamic acids. Linkages with sulfonic acid reactive groups are alsopossible with the above coupling moiety s and result insulfonylhydrazides or N-sulfonylhydroxylamines. The resulting linkagecan be subsequently stabilized by chemical reduction. For instance, thecarbonyl group reacts readily with hydrazides, aminooxy, andsemicarbazides under mild conditions in aqueous solution, and formshydrazone, oxime, and semicarbazone linkages, respectively, which arestable under physiological conditions. See, e.g., Jencks, W. P. (1959)J. Am. Chem. Soc. 81, 475-481; Shao, J. & Tam, J. P. (1995) J. Am. Chem.Soc. 117:3893-3899.

A native or mutated glycosyltransferase can be employed to transfer amonosaccharide labeling agent containing an azide reactive group, analkyne reactive group, a nitril oxide reactive group, or a diazoalkanereactive group, onto the target glycosyl group (e.g., a glycosyl groupcomprising a fucose linked to a galactose). Once incorporated, theazide, alkyne, nitril oxide, or diazoalkane reactive group on thesaccharide labeling agent can then be modified by, e.g., a Huisgen [3+2]cycloaddition reaction in aqueous conditions in the presence of acatalytic amount of copper (See, e.g., Tornoe, et al., (2002) Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599; Padwa, A. in Comprehensive Organic Synthesis, Vol. 4,(1991) Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen,R. in 1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley,New York, p. 1-176). In a [3+2] cycloaddition addition reaction, whereeither an azide, alkyne, nitril oxide, or diazoalkane is a reactivegroup, the other functionality would act as a coupling moiety. The [3+2]cycloaddition addition reaction can be used to introduce affinity probes(biotin), dyes, polymers (e.g., poly(ethylene glycol) or polydextran) orother monosaccharides (e.g., glucose, galactose, fucose, 0-GlcNAc,mannose-derived saccharides bearing the appropriate reactive group). TheHuisgen 1,3-dipolar cycloaddition of azides and acetylenes can give1,2,3-triazoles, also called “click chemistry.” (see Lewis W G, Green LG, Grynszpan F, Radic Z, Carlier P R, Taylor P, Finn M G, Sharpless K B.Angewandte Chemie—Int'l Ed. 41 (6): 1053.). In addition,strain-promoted, Cu-free reactions of azides with cyclooctynes anddibenzocyclooctynes can also be used (See, e.g., Jewett and Bertozzi,Chem. Soc. Rev., 2010, 39, 1272-1279; Coats, et al., Org. Lett.7:1469-1472, 2005; Seo, et al., J. Org. Chem. 68:609-612, 2003; Li, etal., Tet. Lett., 45:3143-3146, 2004).

An exemplary method disclosed herein involves a cycloaddition ratherthan a nucleophilic substitution reaction, modification of proteins canbe performed with extremely high selectivity (as opposed to reactionswith amines, carboxylates or sulfhydryl groups which are found morecommonly on the surface of proteins). The reaction can be carried out atroom temperature in aqueous conditions with excellent regioselectivity(1,4>1,5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tomoe, et al., (2002) Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. The resulting five-membered ring that is attached to thelabeling agent and the detection agent that results from the Huisgen[3+2] cycloaddition is not generally reversible in reducing environmentsand is stable against hydrolysis for extended periods in aqueousenvironments.

The reactive group also can be an azido group capable of reacting in aStaudinger reaction (see, for example, Saxon, E.; Luchansky, S. J.;Hang, H. C.; Yu, C.; Lee, S. C.; Bertozzi, C. R.; J. Am. Chem. Soc.;2002; 124(50); 14893-14902.). The Staudinger reaction, which involvesreaction between trivalent phosphorous compounds and organic azides(Staudinger et al. Helv. Chim. Acta 1919, 2, 635), has been used for amultitude of applications. (Gololobov et al. Tetrahedron 1980, 37, 437);(Gololobov et al. Tetrahedron 1992, 48, 1353). There are almost norestrictions on the nature of the two reactants. The phosphine can havea neighboring acyl group such as an ester, thioester or N-acyl imidazole(i.e. a phosphinoester, phosphinothioester, phosphinoimidazole) to trapthe aza-ylide intermediate and form a stable amide bond upon hydrolysis.The phosphine can also be typically a di- or triarylphosphine tostabilize the phosphine.

The labeling agent can comprise an olefin reactive group and can bereacted with a coupling moiety on a detection agent using a crossmetathesis reaction in the presence of a catalyst. In a cross metathesisreaction, where the reactive group is an olefin, a coupling moiety is anolefin, an alkyne, or an appropriate substrate for a metathesis reactionwith an olefin. Commonly, where the reactive group is an olefin, acoupling moiety is also an olefin. Catalysts for a cross metathesisreaction are well-known and include water-soluble catalysts. such asthose described in Lynn D M, Mohr B, Grubbs R H, Henling L M, and Day MW (2000) J. Am. Chem. Soc.; 2000; 122: 6601-6609 and those review inChen L Y, Yang H J, Sun W H (2003) Progress In Chemistry 15: 401-408.

The reactive group is substantially not reactive with components of abiological mixture, such as a typical cellular extract, including forexample, nucleic acids and proteins. An exemplary reactive group is acarbonyl reactive group, which can react with a coupling moiety, such asan aminoxy, hydrazide or thiosemicarbazide group on the detection agent.Another exemplary reactive group is an azide group, which can react witha coupling moiety, such as an alkene group or an alkyne group in areaction, e.g., a Huisgen [3+2] cycloaddition reaction.

In some embodiments, the labeling agent is a UDP-Gal having asubstituent R appeneded at any suitable position of the galactose ring.For example, the substituent R can be appended at C-2, C-3, C-4, or C-6position of the galactose ring. In some embodiments, the labeling agenthas a formula of

In some embodiments, the labeling agent has a formula of

In some embodiments, the labeling agent has a formula of

In some embodiments, the substituent R was appended at the C-2 positionof the galactose ring because the glycosyltransferase of the presentinvention have been shown to tolerate unnatural substrates containingminor substitutions at the C-2 position. In some embodiments, thelabeling agent has a formula I:

In some embodiments, R is a substituent selected from the groupconsisting of straight chain or branched C₁-C₁₂ carbon chain bearing acarbonyl group, azide group, straight chain or branched C₁-C₁₂ carbonchain bearing an azide group, straight chain or branched C₁-C₁₂ carbonchain bearing an alkyne, and straight chain or branched C₁-C₁₂ carbonchain bearing an alkene. In some embodiments, R is selected from thegroup consisting of straight chain or branched C₂-C₄ carbon chainbearing a carbonyl group, azide group, straight chain or branched C₂-C₄carbon chain bearing an azide group, straight chain or branched C₂-C₄carbon chain bearing an alkyne, and straight chain or branched C₂-C₄carbon chain bearing an alkene.

In some embodiments, R is —OH, —NH₂, —NHC(O)CH₃, —NHC(O)—C₁₋₆alkyl,—NHC(O)CH₂N₃, —NHC(O)—C₁₋₆alkyl-N₃, —NHC(O)CH₂C≡CH, or—NHC(O)—C₁₋₆alkyl-C≡CH. In some embodiments, R is not —OH.

In some embodiments, R is optionally substituted aliphatic. In certainembodiments, R is —CH2C(O)R². In certain embodiments, R is —C(O)R². Incertain embodiments, R is —C(O)CH₃, —CH2C(O)CH₃, —C(O)CH═CH₂,—CH₂C(O)CH═CH₂, —C₁₋₆alkyl-C(O)CH═CH₂, —NHC(O)CH═CH₂, —CH₂NHC(O)CH═CH₂,—C₁₋₆alkyl-NHC(O)CH═CH₂, —NHS(O)CH═CH₂, —CH₂NHS(O)CH═CH₂, or—C₁₋₆alkyl-NHS(O)CH═CH₂. In some embodiments, R is —(CH₂)q-C(O)R²,wherein q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, R² is aliphaticoptionally substituted with —N₃, —CN, —NC, —NCO, —OCN, —NCS, —SCN, —NO,or —N₂. In certain embodiments, R is —(CH₂)qC(O)CH₂N₃, —(CH₂)qC(O)CH₂CN,—(CH₂)qC(O)CH2NC, —(CH₂)qC(O)CH₂OCN, —(CH₂)qC(O)CH₂NCO,—(CH₂)qC(O)CH₂NCS, —(CH₂)qC(O)CH₂SCN, —(CH₂)qC(O)CH₂NO, or—(CH₂)qC(O)CHN₂. In certain embodiments, R is —C(O)CH₂N₃, —C(O)CH₂CN,—C(O)CH₂NC, —C(O)CH₂OCN, —C(O)CH₂NCO, —C(O)CH₂NCS, —C(O)CH₂SCN,—C(O)CH₂NO, or —C(O)CHN₂, wherein q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In certainembodiments, R is —CH₂C(O)CH₂N₃, —CH₂C(O)CH₂CN, —CH₂C(O)CH₂NC,—CH₂C(O)CH₂OCN, —CH₂C(O)CH₂NCO, —CH₂C(O)CH₂NCS, —CH₂C(O)CH₂SCN,—CH₂C(O)CH₂NO, or —CH₂C(O)CH₂N₂. In some embodiments, R² is optionallyaliphatic substituted with halo, e.g., aliphatic substituted withfluoro, chloro, bromo, or iodo. In certain embodiments, R is—(CH₂)qC(O)CH₂Cl, —(CH₂)qC(O)CH₂Br, or —(CH₂)qC(O)CH₂I, wherein q is 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, or 24. In certain embodiments, R is —C(O)CH₂C1, —C(O)CH₂Br,or —C(O)CH₂I. In certain embodiments, R is —CH₂C(O)CH₂C1, —CH₂C(O)CH₂Br,or —CH₂C(O)CH₂I. In certain embodiments, R is —(CH₂)qC(O)CH₂CF₃, whereinq is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, or 24. In certain embodiments, R is —C(O)CH₂CF₃. Incertain embodiments, R is —CH₂C(O)CH₂CF₃. In some embodiments, R² isoptionally aliphatic substituted with amino. In certain embodiments, Ris —(CH₂)qC(O)CH₂NH₂, wherein q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In certainembodiments, R is —C(O)CH₂NH₂. In certain embodiments, R is—CH₂C(O)CH₂NH₂. In certain embodiments, R² is optionally aliphaticsubstituted with hydroxy. In certain embodiments, R is —(CH₂)qC(O)CH₂OH,wherein q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, or 24. In certain embodiments, R is—C(O)CH₂OH. In certain embodiments, R is —CH₂C(O)CH₂OH. In certainembodiments, R² is optionally aliphatic substituted with aryl orheteroaryl. In certain embodiments, R² is optionally —CH₂-aryl or—CH₂-heteroaryl. In certain embodiments, R² is optionally aliphaticsubstituted with optionally substituted heterocyclyl.

In certain embodiments, R is —N(R³)2. In certain embodiments, R is —NH2.In certain embodiments, R is —NHC(O)R². In certain embodiments, R is—NHC(O)R², wherein R² is optionally substituted aliphatic. In certainembodiments, R is —NHC(O)CH₃, or —NHC(O)CH═CH₂. In certain embodiments,R is —NHC(O)R², wherein R² is aliphatic substituted with halo. Incertain embodiments, R is —NHC(O)R², wherein R² is aliphatic substitutedwith chloro, bromo, or iodo. In certain embodiments, R is —NHC(O)R²,wherein R² is aliphatic substituted with fluoro. In certain embodiments,R is —NHC(O)CH₂C1, —NHC(O)CH₂Br, or —NHC(O)CH₂I. In certain embodiments,R is —NHC(O)CH₂CF₃. In certain embodiments, R is —NHC(O)R², wherein R²is aliphatic substituted with —N₃, —CN, —NC, —NCO, —OCN, —NCS, —SCN,—NO, or —N₂. In certain embodiments, R is —NHC(O)CH₂N₃, —NHC(O)CH₂CN,—NHC(O)CH₂NC, —NHC(O)CH₂OCN, —NHC(O)CH₂NCO, —NHC(O)CH₂NCS,—NHC(O)CH₂SCN, —NHC(O)CH₂NO, or —NHC(O)CHN₂. In certain embodiments, Ris —NHC(O)R², wherein R² is aliphatic substituted with amino. In certainembodiments, R is —NHC(O)CH₂NH₂. In certain embodiments, R is —NHC(O)R²,wherein R² is aliphatic substituted with hydroxy. In certainembodiments, R is —NHC(O)CH₂OH. In certain embodiments, R is —NHC(O)R²,wherein R² is aliphatic substituted with aryl or heteroaryl. In certainembodiments, R is —NHC(O)R², wherein R² is —CH₂-aryl or —CH₂heteroaryl.In certain embodiments, R is —NHC(O)R², wherein R² is aliphaticsubstituted with optionally substituted heterocyclyl.

In certain embodiments, R is —OR⁴. In certain embodiments, R is —OH. Incertain embodiments, R is —O— (protecting group). In certainembodiments, R is —OAc. In certain embodiments, R is —OC(O)R². Incertain embodiments, R is —OC(O)R², wherein R² is optionally substitutedaliphatic. In certain embodiments, R is —OC(O)CH₃, or —OC(O)CH═CH₂. Incertain embodiments, R is —OC(O)R², wherein R² is aliphatic substitutedwith halo. In certain embodiments, R is —OC(O)R², wherein R² isaliphatic substituted with chloro, bromo, or iodo. In certainembodiments, R is —OC(O)R², wherein R² is aliphatic substituted withfluoro. In certain embodiments, R is —OC(O)CH₂Cl, —OC(O)CH₂Br, or—OC(O)CH₂I. In certain embodiments, R is —OC(O)CH2CF. In certainembodiments, R is —OC(O)R², wherein R² is aliphatic substituted with—N3, —CN, —NC, —NCO, —OCN, —NCS, —SCN, —NO, or —N₂. In certainembodiments, R is —OC(O)CH₂N₃, —OC(O)CH₂CN, —OC(O)CH₂NC, —OC(O)CH₂OCN,—OC(O)CH₂NCO, —OC(O)CH₂NCS, —OC(O)CH₂SCN, —OC(O)CH₂NO, or —OC(O)CHN₂. Incertain embodiments, R is —OC(O)R², wherein R² is aliphatic substitutedwith amino. In certain embodiments, R is —OC(O)CH₂NH₂. In certainembodiments, R is —OC(O)R², wherein R² is aliphatic substituted withhydroxy. In certain embodiments, R is —OC(O)CH₂OH. In certainembodiments, R is —OC(O)R², wherein R² is aliphatic substituted witharyl or heteroaryl. In certain embodiments, R is —OC(O)R², wherein R² is—CH₂-aryl or —CH₂-heteroaryl. In certain embodiments, R is —OC(O)R²,wherein R² is aliphatic substituted with optionally substitutedheterocyclyl.

In some embodiments, the labeling agent has a formula II:

In some embodiments, the labeling agent has a formula III:

A labeling agent has a formula II was synthesized from the previouslyreported synthesis scheme (Hang, H. C.; Bertozzi, C. R. J. Am. Chem.Soc. 2001, 123, 1242-1243). The following conditions can be used: (a)Me₂NH, THF (53%); (b) (BnO)₂PNiPr₂, then mCPBA (54%); (c) Pd/C, H₂,tri-n-octylamine; (d) UMP-morpholidate, 1H-tetrazole, pyr; (e) TEA,H₂O/MeOH (45%, 3 steps). Synthesis of a agent has a formula I followsclosely this scheme, except with the use a different starting material.

A variety of detection agents can be used. The detection agent canitself be detectable, or can be used to recruit another labelingmolecule or enzyme, a secondary detection agent. The detection agent hasa coupling moiety that can bind to or react with the reactive group.

A detection agent is an agent that has a property that can be observedspectroscopically or visually. Methods for production of detectablylabeled proteins using detection agents are well known in the art. Thedetection agent can be detectable through various detection means, suchas radioactively, chemiluminescence, fluorescence, mass spectrometry,spin labeling, affinity labeling, or the like. The detection agent alsocan be detectable indirectly, for example, by recruitment of one or moreadditional factors.

A radioactive substance refers to a radioactive atom, a substance havingradioactive atoms incorporated therein, or a substance radiolabeled withan additional or substituted radioactive atom not normally found in thenative substance. Examples of radioactive atoms include, but are notlimited to, ³²P, ³³P, ³⁵S, 125I, ³H, ¹³C, ¹⁴C, ⁵¹Cr and ¹⁸O. In oneembodiment, the reactive group further comprises such a radioactivesubstance.

Most chemiluminescence methods involve chemical components to actuallygenerate light. Chemiluminescence is the generation of electromagneticradiation as light by the release of energy from a chemical reaction.While the light can, in principle, be emitted in the ultraviolet,visible or infrared region, those emitting visible light are the mostcommon. Chemiluminescent reactions can be grouped into three types:

1) Chemical reactions using synthetic compounds and usually involving ahighly oxidized species, such as peroxide, are commonly termedchemiluminescent reactions.

2) Light-emitting reactions arising from a living organism, such as thefirefly or jellyfish, are commonly termed bioluminescent reactions.

3) Light-emitting reactions which take place by the use of electricalcurrent are designated electrochemiluminescent reactions.

Examples of chemiluminescent detection agents include, but are notlimited to, luminol chemiluminescence, peroxyoxalate chemiluminescence,and diphenylanthracene chemiluminescence.

Fluorescence is the phenomenon in which absorption of light of a givenwavelength by a fluorescent molecule is followed by the emission oflight at longer wavelengths. Examples of fluorescent detection agentsinclude, but are not limited to, rhodamine, fluorescein, Texas red,cyanine dyes, nanogold particles coated with gold, and analogues thereofand alike.

Mass spectrometry is an analytical technique that is used to identifyunknown compounds, quantify known materials, and elucidate thestructural and physical properties of ions. Mass Spectrometry can beused in conjunction with chromatography techniques, such as LC-MS andGC-MS. Examples of mass spectrometry tools for use as detection agentsinclude, but are not limited to, electron ionisation (EI), chemicalionisation (CI), fast atom bombardment (FAB)/liquid secondary ionisation(LSIMS), matrix assisted laser desorption ionisation (MALDI), andelectrospray ionisation (ESI). See, for example, Gary Siuzdak, MassSpectrometry for Biotechnology, Academic Press, San Diego, 1996.

Electron paramagnetic resonance (EPR), also known as electron spinresonance (ESR) and electron magnetic resonance (EMR), is the name givento the process of resonant absorption of microwave radiation byparamagnetic ions or molecules, with at least one unpaired electronspin, and in the presence of a static magnetic field. Species thatcontain unpaired electrons include free radicals, odd electronmolecules, transition-metal complexes, lanthanide ions, andtriplet-state molecules.

Affinity labeling is a method for tagging molecules so that they can bemore easily detected and studied. Affinity labeling can be based onsubstituting an analogue of a native substrate.

In one embodiment, the detection agent is a biotin or a biotinderivative. Biotin and biotin derivatives are well known to one of skillin the art, and are described in the Handbook of Fluorescent Probes andResearch Products, Ninth Edition, Molecular Probes, Eugene, Oreg., 2002.Additional detection schemes also are provided in the Handbook.Secondary detection agents also are disclosed, including fluorescentreagents (e.g., fluorescently labeled streptavidin) and enzymaticreagents that can convert substrates colorimetrically orfluorometrically (e.g., streptavidin alkaline phosphatase andstreptavidin-horseradish peroxidase conjugates). A number of detectionschemes are known to one of skill in the art and include, for example:fluorescent and luminescent probes (e.g., fluoroscein hydrazide, metalnanoparticles or quantum dots) (see, e.g., Geoghegan, K. F. & Stroh, J.G. (1992) Bioconjug. Chem. 3:138-146); metal-binding probe (e.g.,polyhistidine tag or metal chelate); protein-binding probes (e.g.,FLAG-tag); probe (e.g., dinitrophenol) for antibody-based binding;radioactive probe (circumvent challenging synthesis and handling ofradiolabeled monosaccharides); photocaged probe; spin-label orspectroscopic probe; heavy-atom containing probe (i.e., Br, I) for x-raycrystallography studies; polymer (e.g. PEG- or poly(propylene)glycol)containing probe; probes that permit protein cross-linking (e.g., tocovalently modify binding partners to protein being modified, such ascontaining diazirene, benzophenone, or azidophenyl groups); and bindingto particles or surfaces that contain complementary functionality.

In some embodiments, the present invention provide methods for the rapidand sensitive detection of fucose-α(1-2)-galactose group containingglycans (e.g., glycoproteins). One approach capitalizes on the substratetolerance of glycosyltransferases of the present invention, which allowsfor chemoselective installation of a non-natural reactive group (e.g., aketone or azide reactive group) to fucose-α(1-2)-galactose groupcontaining proteins (FIG. 1A). These reactive groups (e.g., a ketone orazide reactive group) have been well-characterized in cellular systemsas a neutral, yet versatile reactive group. In some embodiments, theketone or azide reactive group serves as a unique marker to “tag”fucose-α(1-2)-galactose group containing proteins with biotin. Oncebiotinylated, the modified proteins can be readily detected byfluorescence or chemiluminescence, such as using streptavidin conjugatedto horseradish peroxidase (HRP).

FIG. 1A shows a general strategy for detection offucose-α(1-2)-galactose group containing proteins. In some exemplaryembodiments, as shown in FIG. 1A, the methods are used to detectfucose-α(1-2)-galactose moiety on a protein or a mixture of proteins.According to the methods, a protein having the fucose-α(1-2)-galactosemoiety is contacted with a labeling agent comprising a reactive group.The labeling agent can be a substrate of a particular enzyme that reactswith the fucose-α(1-2)-galactose moiety on the protein to be labeled,for example, the labeling agent can be an analog of uridyl phosphatesugar. A glycosyltransferase can transfer the labeling agent to thefucose-α(1-2)-galactose pendant moiety on the protein. In oneembodiment, the reactive group is a ketone or azide moiety, which issubstantially unreactive with biological constituents. When the reactivegroup is a ketone, the labeled protein can then be reacted with adetection agent comprising a coupling moiety, for example, a detectionagent having an aminoxy, hydrazide or thiosemicarbazide coupling moiety.When the reactive group is an azide, the labeled protein can then bereacted with a detection agent comprising a coupling moiety, forexample, via a Huisgen [3+2] cycloaddition reaction. The detection agentcan be a biotin moiety, which allows recruitment of a variety of avidin-or streptavidin-linked secondary detection agents, including fluorescentdyes and enzymes that can convert substrates to give a detectablesignal.

In one embodiment, the present method is applied to label or detect aglycan in a biological sample. Exemplary biological samples includetissue samples or bodily fluid samples. The sample can be a tumor biopsysample, a blood, plasma, or serum sample, lymphatic fluid, saliva, alung aspirate, a nipple aspirate, breast duct lavage sample, a pelviclavage sample, a swab or scraping, etc. In some embodiments, the samplesinclude whole cells (e.g., tumor cells or blood cells).

In one embodiment, the detection agent is a biotin moiety. When thedetection agent is a biotin moiety, it can be used to noncovalentlyrecruit a number of secondary detection agents, including, for example,enzymes capable of making reacting with fluorogenic, chemiluminescent,calorimetric products. The biotin is also useful for affinitychromatography using streptavidin/avidin conjugated tosepharose/agarose. Affinity enrichment allows for the enrichment ofglycopeptides present in low cellular abundance. Fucose-α(1-2)-galactosecontaining peptides can be challenging to detect by mass spectrometry inthe absence of enrichment strategies. According to some embodiments,biological mixtures, such as cell lysates, can be labeled with thelabeling agent having a formula I, II or III. Such biological mixturescan then be: digested with protease such as trypsin, capturedglycopeptides using monomeric avidin conjugated to agarose, eluted theglycopeptides and identified the peptides by LC-MS. Accordingly, aprotein having a fucose-α(1-2)-galactose moiety in a nuclear lysate, canbe labeled using the methods of the present invention with a ketone orazide reactive group-containing labeling agent and reacted with a biotinderivative. The labeled protein can then be detected by blotting withstreptavidin-HRP. Such procedures can allow for high-throughputidentification of the fucose-α(1-2)-galactose proteome. Anotheradvantage of the streptavidin-agarose is that intact glycoproteins canbe isolated. This procedure can be useful for rapid and fairlyhigh-throughput detection by Western blotting (e.g., label proteins,isolate fucose-α(1-2)-galactose glycosylated proteins, and then probethe Western blot with antibodies against proteins of interest. Thisprocedure can circumvent developing ways to immunoprecipitate or purifyeach protein of interest.). This procedure can also be used inconjunction with chromatin immunoprecipitation (CHIP assays) protocolsto identify the genes regulated by post-translationally modifiedtranscription factors.

One approach capitalizes on the substrate tolerance ofglycosyltransferases, which allows for chemoselective installation of anon-natural functionality, such as a ketone or azide reactive group, tofucose-α(1-2)-galactose moiety on modified proteins (FIG. 1A).

Human blood group A antigen glycosyltransferase or bacteria homologuethereof has been shown to tolerate unnatural substrates containingsubstitutions at one or more positions on the sugar ring (e.g., the C-2position). Enzyme design to enlarge binding pockets to accommodatealtered substrates for these glycosyltransferases is contemplated.Generally, the binding pocket for the glycosyltransferase is identified,for instance, through crystal structure analysis. Then, the individualresidues of the binding pocket of the glycosyltransferase can bemutated. Through homology modeling, the binding pocket of the mutatedglycosyltransferase can be envisioned. Further modeling studies canexplore binding of substrates in the binding pocket of the mutatedglycosyltransferase. An exemplary mutated enzyme would enlarge thebinding pocket of the enzyme and/or enhance the catalytic activitytoward substrates without compromising specificity.

In general, a novel chemoenzymatic strategy that detects a glycosylgroup comprising a fucose linked to a galactose with a high efficiencyand sensitivity is disclosed. A variety of applications, includingdirect fluorescence detection, affinity enrichment, and isotopiclabeling for comparative proteomics, is also contemplated.

The present invention therefore provides a labeled protein obtained bythe methods described above. For example, a composition comprising alabeled protein substantially free of unlabeled protein can be made fromthe processes described in the specification.

Specifically, in some embodiments, the labeled protein comprises twoglycosyl groups linked together. The first glycosyl group is a glycosylgroup comprising a fucose linked to a galactose. The second glycosylgroup is a glycosyl group covalently linked to the first glycosyl group.The second glycosyl group can be covalently linked to the first glycosylgroup at any positions. For example, the second glycosyl group can becovalently linked to the fucose group on the first glycosyl group, orvia the galactose on the first glycosyl group. In some embodiments, thefirst glycosyl group is covalently linked to the first glycosyl group atC-3 position of galactose on the first glycosyl group.

The second glycosyl group can be any sugar suitable for covalentlyconjugating with the first glycosyl group. In some embodiments, thesecond glycosyl group comprises a reactive group. In some embodiments,the second glycosyl group is a modified galactose, e.g., a GalNac. Insome embodiments, the labeled protein has a formula of

wherein R is a substituent comprising a reactive group, e.g., carbonylgroup, azide group, nitril oxide group, diazoalkane group, alkyne group,and olefin group. In some embodiments, the labeled protein has a formulaof

In some embodiments, the labeled protein has a formula of

In some embodiments, the labeled protein further comprises a detectionagent covalently linked to the second glycosyl group. For example, thedetection agent can be covalently linked to the second glycosyl groupvia a reaction between the reactive group on the second glycosyl groupand a coupling moiety on the detection agent. In some embodiments, thedetection agent is biotin or biotin derivative. In some embodiments, thelabeled protein has a formula of

Y is the detection agent covalently linked to the second glycosyl groupvia the reaction between the reactive group on the second glycosyl groupand the coupling moiety on the detection agent. In some embodiments, thelabeled protein has a formula of

In some embodiments, the labeled protein has a formula of

In some embodiments, the labeled protein further comprises an additionalagent recruited by the detection agent as described herein. Exemplaryadditional agents include a secondary labeling agent, an enzyme, and asecondary detection agent. The additional agent can be non-covalentlyattached or covalently linked to the detection agent.

The present invention also provides a reaction mixture comprising (1) aglycan (e.g., a glycoprotein) with a glycosyl group comprising a fucoselinked to a galactose, and (2) a labeling agent comprising atransferable glycosyl group recognized by a transferase capable oftransferring the transferable glycosyl group to the glycan. In someembodiments, the transferable glycosyl group comprises a reactive group.In some embodiments, the reactive group is capable of reacting with acoupling moiety on a detection agent to form a covalent bond.

In some embodiments, the mixture further comprises a detection agent asdescribed herein. In some embodiments, the detection agent comprises acoupling moiety that is capable of reacting with the reactive group onthe labeling agent to form a covalent bond.

In some embodiments, the mixture further comprises a glycosyltransferaseas described herein. In some embodiments, the glycosyltransferase isspecific for a glycosyl group comprising a fucose linked to a galactoseand is capable of catalyzing the transfer of the transferable glycosylgroup on the labeling agent to the glycosyl group comprising a fucoselinked to a galactose. In some embodiments, the glycosyltransferase is aglycosyltransferase specific for a fucose-α(1-2)-galactose group. Forexample, the glycosyltransferase can be a human blood group A antigenglycosyltransferase or a variant or fragment thereof, e.g., a bacteriahomologue of the human blood group A antigen glycosyltransferase (BgtA)or a variant or fragment thereof.

In some embodiments, the mixture further comprises a secondary agent asdescribed herein. For example, the secondary agent can be a secondarylabeling agent, an enzyme, or a secondary detection agent. In someembodiments, the secondary agent is an agent that can be recruited bythe detection agent. Exemplary secondary detection agents includefluorescent reagent, enzymatic reagent capable of converting substratescolorimetrically or fluorometrically, fluorescent and luminescent probe,metal binding probe, protein-binding probe, probe for antibody-basedbinding, radioactive probe, photocaged probe, spin-label orspectroscopic probe, heavy-atom containing probe, polymer containingprobe, probe for protein cross-linking, and probe for binding toparticles or surfaces that contain complementary functionality.

The present invention further provides a kit for labeling (e.g.,detecting) a glycan (e.g., a glycoprotein) with a glycosyl groupcomprising a fucose linked to a galactose as described herein. In someembodiments, the kit comprises a glycosyltransferase specific for aglycosyl group comprising a fucose linked to a galactose, e.g., aglycosyltransferase is specific for a fucose-α(1-2)-galactose group. Theglycosyltransferase is capable of catalyzing the transfer of a labelingagent to the glycan. Glycosyltransferases useful for the presentinvention are described herein. Exemplary glycosyltransferases include ahuman blood group A antigen glycosyltransferase or a variant or fragmentthereof, e.g., a bacteria homologue of the human blood group A antigenglycosyltransferase (BgtA) or a variant or fragment thereof. In someembodiments, the kit further comprises instructions instructing a userto perform the detection. In some embodiments, the kit can be used fordetecting cancer.

In some embodiments, the kit further comprises a labeling agent asdescribed herein. In some embodiments, the labeling agent is an agentcomprising a reactive group and a transferable glycosyl group recognizedby the transferase. Where desired, the reactive group is located on thetransferable glycosyl group.

In some embodiments, the kit further comprises a detection agent asdescribed herein. In some embodiments, the detection agent comprises acoupling moiety capable of reacting with the reactive group on thelabeling agent to form a covalent bond. Exemplary detection agentsinclude fluorescent reagent, enzymatic reagent capable of convertingsubstrates calorimetrically or fluorometrically, fluorescent andluminescent probe, metal-binding probe, protein-binding probe, probe forantibody-based binding, radioactive probe, photocaged probe, spin-labelor spectroscopic probe, heavy-atom containing probe, polymer containingprobe, probe for protein cross-linking, and probe for binding toparticles or surfaces that contain complementary functionality.

In some embodiments, the kit further comprises a storage buffer. Thestorage buffer can comprise a stabilizer such as bovine serum albumin,gelatin, glycerol, sodium azide, tris, or a combination thereof.

In some embodiments, the kit further comprises a separation device forpurifying a target glycan (e.g., a target glycoprotein) before labeling,and/or for purifying a labeled target glycan. Methods for purifyingglycan (e.g., glycoprotein) are known in the art, non-limiting examplesof which include protein A-based affinity chromatography, size-exclusionchromatography, and ultra membrane filtration. Exemplary separationdevices include biotin affinity chromatography column. Commercial kitsfor protein purification, such as antibody cleaning kits, may also beused for target protein purification. Size-exclusion columnchromatography and ultra membrane filtration are useful for removingstabilizers/preservatives of relatively small molecules, such asglycerol, Tris, and amino acids. Ultra membrane filtration may also beused to adjust target protein concentration to a desired value. Ultramembrane filtration can be conveniently and rapidly carried out on acentrifuge using a commercial ultra membrane filtration vial. Themembranes in such ultra filtration devices typically have different poresizes, or so-called Molecular Weight Cut-off sizes (MWCO), permittingrelatively small molecules to go through while retaining biggermolecules, such as proteins. In some embodiments, a membrane with a MWCOof about, less than about, or more than about 1, 5, 10, 15, 20, 25, 50,100, or more kD is provided to purify a target protein before and/orafter labeling. Columns for size exclusion chromatography typicallycomprise particles or beads having pores of a particular MWCO. Particleslarger than the MWCO pass through the column faster than particles at orbelow the MWCO. In some embodiments, a column with a MWCO of about, lessthan about, or more than about 1, 2, 10, 15, 20, 25, 50, 100, or more kDis provided to purify a target protein before and/or after labeling.

In some embodiments, the kit further comprises a stain stabilizingreagent for enhancing dye fluorescence, such as by enhancing fluorescentintensity or reducing a rate of decrease in fluorescent intensity. Stainstabilizing reagents are known in the art, non-limiting examples ofwhich include EverBrite (Biotium), Vectashield (Vector Laboratories),and SlowFade Gold (Invitrogen). In some embodiments, fluorescentintensity of a dye in the presence of the stain stabilizing reagent isincreased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, 300%, 400%, 500% or more above the intensity of thedye in the absence of the stain stabilizing reagent. In someembodiments, fluorescent intensity of a dye in the presence of the stainstabilizing reagent is maintained above a threshold level for a timethat is at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, or more minutes. In some embodiments, thethreshold level is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more of thestarting fluorescent intensity.

In some embodiments, the kit further comprises a detectable label suchas a radio-opaque label, nanoparticle, PET label, MRI label, radioactivelabel, and the like.

The present invention further provides a method of identifying a glycan(e.g., a glycoprotein) comprising a fucose linked to a galactose. Themethod comprises the steps of 1) providing one or more homogenouspopulation of glycans; 2) contacting a glycosyl transferase with theglycans in the presence of a labeling agent comprising a transferableglycosyl group, wherein the transferable glycosyl group comprises areactive group, wherein the glycosyl transferase is specific for theglycosyl group comprising a fucose linked to a galactose and catalyzesthe transfer of the transferable glycosyl group to the glycosyl groupcomprising a fucose linked to a galactose; 3) contacting the glycanswith a detection agent, wherein the reactive group on the labeling agentis capable of reacting with a coupling moiety on the detection agent toform a covalent bond; and 4) identifying a glycan having the covalentlybound detection agent via the reactive group on the transferred glycosylgroup as the glycan comprising a fucose linked to a galactose.

In some embodiments, the glycans are covalently or non-covalentlyattached to a solid support. In some embodiments, the glycans areattached to the solid support in the form of an array. In someembodiments, the array comprises two or more addressable locations. Eachaddressable location may comprise a homogenous population of glycansdisplaying different carbohydrate sequences.

In some embodiments, arrays of glycans are formed on a substrate. Glycanmolecules can be arranged directly on a substrate for use in, forexample, the identification of one or more glycans, e.g., a glycancomprising a fucose linked to a galactose. The glycan molecules can beattached to the substrate via linkers, using methods well known in theart and as described herein. Alternatively, the glycan molecules can besynthesized directly on the substrate. In some embodiments, glycanmolecules are non-covalently attached to PLL coated substrates, or glassslides. Antibodies to specific glycan molecules can be used to determinethat the array is composed of the desired glycan molecules.

In a typical array a substrate comprises one or more addressablelocations of glycan molecules. The addressable locations can be directlyadjacent to each other or can be physically separated by a gap or abarrier.

In some embodiments, each addressable location comprises one type ofglycan molecule or more than one type of glycan molecules. Glycanmolecules may differ in the length of the oligosaccharide chains or mayalso differ as different subtypes of glycan molecules.

In the case where each location comprises a single type of glycanmolecule, the number of locations on the array of glycan molecules is atleast as great as the number of different types of glycan molecules tobe used. In one embodiment, each addressable location comprises at leastabout 10⁵ glycan molecules.

In some embodiments, the particular location of a particular type ofglycan molecule is not predetermined. The identity of the glycan can bedetermined using the methods described herein. In some embodiments, theglycan molecule composition and physical location of each addressablelocation is known. In one embodiment, each of the addressable locationsin the array comprises a different type of glycan molecule. For example,if a target protein is to be assayed for its ability to bind varioustypes of glycans containing a fucose linked to a galactose, the arraymay comprise multiple addressable locations, each comprising a differenttype of glycan molecule containing a fucose linked to a galactose. In analternative embodiment, more than one addressable location comprising aparticular type of glycan molecule is present.

The overall size of the array is not limited and will be determinedbased on a variety of factors, including the number of glycan moleculesin each addressable locations, the number of addressable locations andthe physical nature of the solid support on which the array is formed.

In some embodiments, a glycan microarray can be created using a general,highly efficient strategy for attaching glycans to the array surface,details of which are provided in Tully, S. E. et al., J. Am. Chem. Soc.(2006) 128:7740-7741; Gama, C. I. et al., Nature Chemical Biology (2006)2(9):467-473; and Shipp, E. L. and Hsieh-Wilson, L. C., Chemistry &Biology (2007) 14:195-208; each of which are incorporated herein byreference in its entirety. Briefly, glycan molecules are synthesizedwith an allyl functionality on the reducing end of the sugar. This groupis stable to the chemical manipulations used to synthesize theoligosaccharides, yet it can be readily functionalized for surfaceconjugation.

In a particular embodiment, solutions of aminooxy oligosaccharides inbuffer (for example, 300 mM NaH₂PO₄, pH 5.0) can be arrayed on slides,such as Hydrogel Aldehyde slides (NoAb Biodiscoveries) by using arobotic arrayer, such as a Microgrid 11 arrayer (Biorobotics), todeliver sub-nanoliter volumes. In one embodiment, for example, spots areapproximately 1 to 1000 μm, in some embodiments 1 to 500 μm and in someembodiments about 100-200 μm in diameter. In some embodiments,concentrations of carbohydrates range from 0-1000 μM. The resultingarrays can be incubated in a 70% humidity chamber at room temperatureovernight and then stored in a low humidity, dust-free dessicator priorto use.

Importantly, this strategy requires minimal manipulation of the glycan,enabling their direct conjugation in two short, high-yielding steps.Moreover, the approach is compatible with standard DNA robotic printingand fluorescence scanning technology, which requires only minimalamounts of material and allows a large number of molecular interactionsto be probed simultaneously.

An exemplary glycan array is the printed glycan array provided by theConsortium for Functional Glycomics (www.functionalglycomics.org). Theprinted array uses a library of natural and/or synthetic glycans printedon to glass microscope slides. One or more glycosyl transferases,labeling agents, and detection agents can be added to the array inconditions where the transferable glycosyl group on the labeling agentmay be transferred to the glycan having a glycosyl group comprising afucose linked to a galactose. Other glycan arrays include for examplethe glycan array discussed in Liang et al. 2009 (Expert Rev Proteomics.2009 December; 6(6):631-45) and Blixt et al. 2004 (PNAS 2004 101(49): pp17033-17038), the contents of which are hereby incorporated byreference.

The glycan array may include at least one positive and at least onenegative control. In some embodiments the glycan array may also includeat least one background control. The use of positive, negative andbackground controls in arrays is well known in the art. A negativecontrol is known to give a negative result. The negative control may bea molecule which is known not to have a glycosyl group comprising afucose linked to a galactose. Alternatively the negative control may becreated by the absence of a glycan bound to the solid support, i.e., sothere is nothing for a candidate antibody to bind. The positive controlconfirms that the basic conditions of the experiment were able toproduce a positive result. The positive control may be a molecule, e.g.a glycan, which is known to a glycosyl group comprising a fucose linkedto a galactose (e.g., a fucose-α(1-2)-galactose group).

The present invention further provides a method of detecting cancercells. The method comprises the steps of 1) contacting a cell with aglycosyltransferase and a labeling agent comprising a transferableglycosyl group as described herein, wherein the transferable glycosylgroup comprises a reactive group, wherein the glycosyltransferase isspecific for the glycosyl group comprising a fucose linked to agalactose and catalyzes the transfer of the transferable glycosyl groupto the glycosyl group comprising a fucose linked to a galactose; 2)contacting the cell comprising the transferred labeling agent with adetection agent, wherein the reactive group on the labeling agent reactswith a coupling moiety on the detection agent to form a covalent bond;and 3) detecting the amount of the detection agent covalently bound tothe cell via the reactive group on the transferred labeling agent.

In some embodiments, the method further comprises 4) comparing theamount of the detection agent covalently bound to the cell to the amountof the detection agent covalently bound in a non-cancerous control. Anincrease (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300% or more) in the amount of the detection agent covalently bound tothe cell from the tissue sample as compared to the amount of thedetection agent covalently bound in a non-cancerous control indicates apresence of cancer cell having the glycosyl group comprising a fucoselinked to a galactose. An amount of the detection agent covalently boundto the cell from the tissue sample that is comparable to the amount ofthe detection agent covalently bound in a non-cancerous controlindicates an absence of cancer cell having the glycosyl marker groupcomprising a fucose linked to a galactose.

In some embodiments, the method further comprises 4) comparing theamount of the detection agent covalently bound to the cell to the amountof the detection agent covalently bound in a cancerous positive control.An amount of the detection agent covalently bound to the cell from thetissue sample that is comparable to the amount of the detection agentcovalently bound in a cancerous positive control indicates a presence ofcancer cell having the glycosyl group comprising a fucose linked to agalactose. A decrease (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more) in the amount of the detection agent covalently bound to thecell from the tissue sample as compared to the amount of the detectionagent covalently bound in a cancerous positive control indicates anabsence of cancer cell having the glycosyl group comprising a fucoselinked to a galactose.

Various cancer cells can be detected using the method described herein.The cells can be cultured cell or cells from a tissue sample of asubject. Expression levels of a glycosyl group comprising a fucoselinked to a galactose (e.g., a fucose-α(1-2)-galactose group) are linkedto (e.g., up-regulated) various cancers, e.g., breast cancer (e.g.,highly invasive breast cancer), lung cancer (e.g., small cell lungcancer), prostate cancer, colon cancer, colorectal cancer, cervicalcancer, and pancreatic cancer. Using the method described herein, thesecancer cells can be detected.

The unique glycan biomarker also enables more efficient drug developmentthrough the discovery of protein targets associated with the glycanmarker. In addition, the unique glycan biomarker as described herein canserve as a basis for novel diagnostics, including companion diagnostics.Examples of proteins or lipids associated with fucose-α(1-2) galactosein cancers include prostate specific antigen (PSA), Globo H antigen (aglycosphingolipid upregulated in breast cancer), and CD44v6 (coloncancer).

The present invention further provides diagnostic methods for detectioncancer. In some embodiments, the compositions and methods describedherein can be used for in vivo or ex vivo diagnosis of cancer. For thesediagnostic applications, the labeling agent and/or detection agent cancomprise a detectable label or an epitope tag that is capable of bindingto a detectable label. Suitable detectable labels include, but are notlimited to radio-opaque labels, nanoparticles, PET labels, MRI labels(e.g., gadolinium-containing contrast agents, iron oxide, manganese, andiron platinum), radioactive labels (e.g., C14 label), and the like.Among the radionuclides useful in various embodiments of the presentinvention, gamma-emitters, positron-emitters, x-ray emitters andfluorescence-emitters are suitable for localization, diagnosis and/orstaging, and/or therapy, while beta and alpha-emitters and electron andneutron-capturing agents, such as boron and uranium, also can be usedfor therapy.

The detectable labels can be used in conjunction with an externaldetector and/or an internal detector and provide a means of effectivelylocalizing and/or visualizing cancer cells associated with fucose-α(1-2)galactose. Such detection/visualization can be useful in variouscontexts including, but not limited to pre-operative and intraoperativesettings. Thus, in certain embodiment this invention relates to a methodof intraoperatively detecting and cancers (e.g., breast cancer, lungcancer, prostate cancer, colon cancer, colorectal cancer, cervicalcancer, and pancreatic cancer) in the body of a mammal.

The examples disclosed below illustrated preferred embodiments and arenot intended to limit the scope. It would be obvious to those skilled inthe art that modifications or variations may be made to the preferredembodiments described herein without departing from the teachings of thepresent invention.

Example 1: Chemoenzymatic Probes for Detecting and ImagingFucose-α(1-2)-Galactose Glycans Biomarkers

Here we report a new chemoenzymatic strategy for the rapid, sensitive,and selective detection of Fucα(1-2)Gal glycans. We demonstrate that theapproach is highly selective for the Fucα(1-2)Gal motif, detects avariety of complex glycans and glycoproteins, and can be used to profilethe relative abundance of the motif on live cells, discriminatingmalignant from normal cells. This approach represents a new potentialstrategy for biomarker detection and expands the technologies availablefor understanding the roles of this important class of carbohydrates inphysiology and disease.

Our approach capitalizes on the substrate tolerance of a bacterialglycosyltransferase to covalently tag specific glycans of interest witha non-natural sugar analog. As the reaction proceeds in quantitativeyield, stoichiometric addition of the non-natural sugar can be achieved,affording higher detection sensitivity relative to antibodies, lectins,and metabolic labeling. Although chemoenzymatic approaches have beenreported for two saccharides, O-linked-β-N-acetylglucosamine (O-GlcNAc)((a) Khidekel, N.; Arndt, S.; Lamarre-Vincent, N.; Lippert, A.;Poulin-Kerstien, K. G.; Ramakrishnan, B.; Qasba, P. K.; Hsieh-Wilson, L.C. J. Am. Chem. Soc. 2003, 125, 16162. (b) Clark, P. M.; Dweck, J. F.;Mason, D. E.; Hart, C. R.; Buck, S. B.; Peters, E. C.; Agnew, B. J.;Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2008, 130, 11576.) andN-acetyllactosamine (LacNAc), (Zheng, T.; Jiang, H.; Gros, M.; Sorianodel Amo, D.; Sundaram, S.; Lauvau, G.; Marlow, F.; Liu, Y.; Stanley, P.;Wu, P. Angew. Chem. Int. Ed. 2011, 50, 4113.) this study demonstratesthe first direct detection of complex oligosaccharides, opening up thepotential to track a broad range of physiologically important glycans.

We exploited the bacterial homologue of the human blood group A antigenglycosyltransferase (BgtA), which has been shown to transferN-acetylgalactosamine (GalNAc) from UDP-GalNAc onto the C-3 position ofGal in Fucα(1-2)Gal structures. (Yi, W.; Shen, J.; Zhou, G.; Li, J.;Wang, P. G. J. Am. Chem. Soc. 2008, 130, 14420.) We reasoned that BgtAmight tolerate substitution at the C-2 position of GalNAc, which wouldallow for the selective tagging of Fucα(1-2)Gal with an azido or ketonefunctionality (FIG. 1A). To test the approach, Fucα(1-2)Gal substrate 1was synthesized via reductive amination of 2′-fucosyllactose withp-nitrobenzylamine and sodium cyanoborohydride (FIGS. 1B and 5,Supporting Information (SI)). Indeed, treatment of 1 with BgtA andeither UDP-N-azidoacetylgalactosamine (UDP-GalNAz, 2) orUDP-2-deoxy-2-(acetonyl)-β-D-galactopyranoside (UDP-ketoGal, 3) led tocomplete conversion to the desired products 4 and 5, respectively, after12 h at 4° C., as determined by liquid chromatography-mass spectrometry(LC-MS; FIGS. 1B, 6 and 7, SI). Kinetic analysis revealed an apparentk_(cat)/K_(m) value of 5.7 nM⁻¹ min⁻¹ for UDP-GalNAz, approximately7-fold lower than the value of 40.4 nM⁻¹ min⁻¹ obtained for the naturalUDP-GalNAc substrate (FIG. 8, SI). (Yi, W.; Shen, J.; Zhou, G.; Li, J.;Wang, P. G. J. Am. Chem. Soc. 2008, 130, 14420.) Subsequent reactionwith an aza-dibenzo-cyclooctyne-biotin derivative (ADIBO-biotin, 6; FIG.6, SI) using copper-free click chemistry (3 h, rt) or with theaminooxy-biotin derivative 7 (FIG. 6, SI; 24 h, rt) afforded thebiotinylated products 8 and 9, respectively, in quantitative yield(FIGS. 1B, 6 and 7, SI).

Having demonstrated that BgtA accepts non-natural substrates, weprofiled the glycans detected by BgtA using carbohydrate microarraysfrom the Consortium for Functional Glycomics. ((a) Blixt, O.; Allin, K.;Bohorov, O.; Liu, X.; Andersson-Sand, H.; Hoffman, J.; Razi, N.Glycoconjugate J. 2008, 25, 59. (b) Blixt, O. et al. Proc. Natl Acad.Sci. USA 2004, 101, 17033.) Glycosylation reactions with BgtA andUDP-GalNAz were performed on 611 different glycans simultaneously at 3different time points (0.5, 2 and 12 h). Following reaction withADIBO-biotin, biotinylated glycans were detected using Cy5-conjugatedstreptavidin. Strong fluorescence labeling of Fucα(1-2)Gal structureswas observed within 0.5 h (FIG. 2A). Notably, the top 26 glycans labeledcontained terminal Fucα(1-2)Gal structures, highlighting the specificityof the chemoenzymatic approach. Moreover, ˜91% of the terminalFucα(1-2)Gal containing a free C-3 hydroxyl group on Gal were labeled onthe array, including the H1 (68, 69) and H2 antigens (76, 77), theganglioside Fuc-GM1 (65), and the Globo H antigen (60), a hexasaccharideoverexpressed on breast, lung and prostate tumors ((b) Chang, W.-W.;Lee, C. H.; Lee, P.; Lin, J.; Hsu, C.-W.; Hung, J.-T.; Lin, J.-J.; Yu,J.-C.; Shao, L.-e.; Yu, J.; Wong, C.-H.; Yu, A. L. Proc. Natl Acad. Sci.USA 2008, 105, 11667. (c) Menard, S.; Tagliabue, E.; Canevari, S.;Fossati, G.; Colnaghi, M. I. Cancer Res. 1983, 43, 1295. (d) Zhang, S.;Zhang, H. S.; Cordon-Cardo, C.; Ragupathi, G.; Livingston, P. O. Clin.Cancer Res. 1998, 4, 2669. (e) Miyake, M.; Taki, T.; Hitomi, S.;Hakomori, S.-i. N. Engl. J. Med. 1992, 327, 14.) and associated withpoor prognosis (FIGS. 2A and 9, SI). ((e) Miyake, M.; Taki, T.; Hitomi,S.; Hakomori, S.-i. N. Engl. J. Med. 1992, 327, 14. (f) Colnaghi, M. I.;Da Dalt, M. G.; Agresti, R.; Cattoretti, G.; Andreola, S.; Di Fronzo,G.; Del Vecchio, M.; Verderio, L.; Cascinelli, N.; Rilke, F. InImmunological Approaches to the Diagnosis and Therapy of Breast Cancer;Ceriani, R. L., Ed.; Plenum Publishing: New York, 1987, 21.) A widevariety of linear (e.g., 501, 75, and 60) and branched structures (e.g.,450, 362, and 457) containing the Fucα(1-2)Gal motif were efficientlylabeled (FIGS. 2A and 10, SI). Modifications of the core disaccharide,such as replacing Gal with GlcNAc, or changing the α(1-2) linkage to anα(1-3), α(1-4) or β(1-3) linkage eliminated the enzymatic labeling byBgtA (e.g., 80, 81, and 82; FIG. 10, SI).

Consistent with a previous report, (Yi, W.; Shen, J.; Zhou, G.; Li, J.;Wang, P. G. J. Am. Chem. Soc. 2008, 130, 14420.) BgtA exhibited morerelaxed specificity toward structures appended to the reducing end ofthe Gal residue. Specifically, glycans containing a β(1-3)GalNAc,β(1-3)GlcNAc, β(1-4)GlcNAc, or β(1-4)Glc in this position wereefficiently labeled (e.g., 62, 66, 74, and 78, respectively; FIGS. 2Aand 9, SI). Although moderate structural substitutions of the GlcNAcwere tolerated such as 6-O-sulfation (e.g., 501 and 222; FIG. 2A),branching at this position via α(1-3) or α(1-4) fucosylation of theGlcNAc residue led to weak labeling, as in the case of the Lewis B (61)and Lewis Y (72, 73) antigens, or no appreciable labeling (e.g., 71, and363; FIGS. 9 and 11, SI). Interestingly, we also observed weak labelingof Galβ(1-4)GlcNAc structures on the glycan array (FIG. 10, SI).However, these structures also exhibited high background signal even inthe absence of UDP-GalNAz, and BgtA failed to label p-nitrophenyl2-acetamido-2-deoxy-4-O-(β-D-galactopyranosyl)-β-D-glucopyranoside(Galβ(1-4)GlcNAc-pNP) in solution (2 h, 25° C.), suggesting thatGalβ(1-4)GlcNAc structures are not covalently labeled by BgtA. Together,these studies demonstrate the strong specificity of BgtA forFucα(1-2)Gal structures and the power of glycan microarrays to rapidlyprofile the specificities of glycosyltransferases for the development ofchemoenzymatic detection strategies.

To determine whether the approach could be used to track Fucα(1-2)Galglycoproteins in complex cell lysates, we labeled proteins from ratbrain extracts with BgtA and UDP-GalNAz, followed by Cu(I)-catalyzedreaction with the alkyne-functionalized tetramethyl-6-carboxy-rhodaminedye 10 (alkyne-TAMRA; FIG. 6, SI). We observed strong fluorescencelabeling of Fucα(1-2)Gal glycoproteins, with minimal non-specificlabeling in the absence of BgtA, UDP-GalNAz, or alkyne-TAMRA (FIG. 12,SI). To confirm further the specificity of the reaction, we labeled thelysates with the alkyne-biotin derivative 11 (FIG. 6, SI), captured thebiotinylated proteins using streptavidin resin, and immunoblotted forthe presence of known Fucα(1-2)Gal glycoproteins. ((a) Murrey, H. E.;Gama, C. I.; Kalovidouris, S. A.; Luo, W.-I.; Driggers, E. M.; Porton,B.; Hsieh-Wilson, L. C. Proc. Natl. Acad. Sci. USA 2006, 103, 21. (b)Murrey, H. E.; Ficarro, S. B.; Krishnamurthy, C.; Domino, S. E.; Peters,E. C.; Hsieh-Wilson, L. C. Biochemistry 2009, 48, 7261.) Neural celladhesion molecule (NCAM), synapsin I, and munc18-1 were allchemoenzymatically labeled and detected in the presence, but not in theabsence, of BgtA (FIG. 2B). In contrast, p44 mitogen-associated proteinkinase (p44 MAPK), a protein that has not been shown to be fucosylated,was not detected. Glycosylated synapsin I was also readily observedfollowing overexpression of Flag-tagged synapsin I in HeLa cells,chemoenzymatic labeling of the lysates with alkyne-TAMRA, synapsinimmunoprecipitation, and visualization using an anti-TAMRA antibody(FIG. 2C). Importantly, UEAI lectin affinity chromatography failed topull-down and detect glycosylated synapsin I when performed on the samescale (FIG. 13, SI). Moreover, previous studies have reported that theFucα(1-2)Gal-specific antibody A46-B/B10 does not immunoprecipitateglycosylated synapsin I from the same neuronal lysates. (Murrey, H. E.;Gama, C. I.; Kalovidouris, S. A.; Luo, W.-I.; Driggers, E. M.; Porton,B.; Hsieh-Wilson, L. C. Proc. Natl. Acad. Sci. USA 2006, 103, 21.) Thus,our chemoenzymatic approach enables the highly sensitive detection ofglycoproteins and provides a variety of different enrichment strategiesand readouts for the Fucα(1-2)Gal motif.

We next investigated whether the chemoenzymatic strategy could be usedto image Fucα(1-2)Gal glycans in cells. HeLa cells overexpressingFlag-tagged synapsin I were fixed, permeabilized, and chemoenzymaticallylabeled on coverslip with BgtA and UDP-GalNAz. Cu(I)-catalyzedazide-alkyne cycloaddition (CuAAC) chemistry was then performed using analkyne-functionalized Alexa Fluor 488 dye (12; FIG. 6, SI) to install afluorescent reporter onto the Fucα(1-2)Gal glycans. Strong fluorescencelabeling was observed in cells transfected with synapsin I, and thelabeling showed excellent co-localization with intracellular synapsin Iexpression (FIG. 3A). No labeling of cells was observed in the absenceof BgtA, and only weak labeling of endogenous Fucα(1-2)Gal glycoproteinswas seen in the absence of synapsin I overexpression (FIG. 3A and FIG.14, SI), confirming the specificity of the in situ chemoenzymaticreaction.

As the Fucα(1-2)Gal epitope has been reported to be a useful biomarkerfor cancer progression and prognosis, ((e) Miyake, M.; Taki, T.; Hitomi,S.; Hakomori, S.-i. N. Engl. J. Med. 1992, 327, 14. (f) Colnaghi, M. I.;Da Dalt, M. G.; Agresti, R.; Cattoretti, G.; Andreola, S.; Di Fronzo,G.; Del Vecchio, M.; Verderio, L.; Cascinelli, N.; Rilke, F. InImmunological Approaches to the Diagnosis and Therapy of Breast Cancer;Ceriani, R. L., Ed.; Plenum Publishing: New York, 1987, 21.) the abilityto detect Fucα(1-2)Gal glycan levels on the surface of cancer cellswould facilitate investigations into Fucα(1-2)Gal as a diagnostic orprognostic marker and a therapeutic target for cancer vaccines. However,antibodies and lectins that bind Fucα(1-2)Gal have been shown tocross-react with other sugar epitopes ((a) Manimala, J. C.; Roach, T.A.; Li, Z.; Gildersleeve, J. C. Angew. Chem. Int. Ed. 2006, 45, 3607.(b) Manimala, J. C.; Roach, T. A.; Li, Z.; Gildersleeve, J. C.Glycobiology 2007, 17, 17C.) such as β-linked Fuc (Manimala, J. C.;Roach, T. A.; Li, Z.; Gildersleeve, J. C. Angew. Chem. Int. Ed. 2006,45, 3607.) or recognize an incomplete subset of Fucα(1-2)Gal glycans(Chang, C.-F.; Pan, J.-F.; Lin, C.-N.; Wu, I.-L.; Wong, C.-H.; Lin,C.-H. Glycobiology 2011, 21, 895), indicating the need for moreselective, yet comprehensive, high-affinity detection methods. Wetherefore applied our chemoenzymatic approach to the detection ofFucα(1-2)Gal glycans on live cancer cells. Cells from the human breastadenocarcinoma cell line MCF-7 were chemoenzymatically labeled with BgtAand UDP-GalNAz for 1 h at 37° C. After reaction with ADIBO-biotin (1 h,rt), Fucα(1-2)Gal glycans were detected using streptavidin conjugated toAlexa Fluor 488 dye. Membrane-associated fluorescence was observed forcells treated with both BgtA and UDP-GalNAz, whereas no labeling wasdetected for control cells labeled in the absence of BgtA (FIG. 3B).

We next compared the expression levels of Fucα(1-2)Gal glycans acrossdifferent cancer and non-cancer cell lines. MCF-7 (breast cancer),MDA-mb-231 (highly invasive breast cancer), H1299 (lung cancer), LnCAP(prostate cancer), and primary prostrate epithelial cells (PrEC) cellswere chemoenzymatically labeled in suspension with BgtA and UDP-GalNAz(2 h, 37° C.), reacted with ADIBO-biotin (1 h, rt), and stained with thestreptavidin-Alexa Fluor 488 conjugate (20 min, 4° C.). As shown by flowcytometry analysis, LnCaP, MCF-7, and MDA-mb-231 cells displayed thehighest levels of fluorescence (FIG. 4), consistent with reports of highGlobo H expression on mammary and prostate tumors. ((b) Chang, W.-W.;Lee, C. H.; Lee, P.; Lin, J.; Hsu, C.-W.; Hung, J.-T.; Lin, J.-J.; Yu,J.-C.; Shao, L.-e.; Yu, J.; Wong, C.-H.; Yu, A. L. Proc. Natl Acad. Sci.USA 2008, 105, 11667. (c) Menard, S.; Tagliabue, E.; Canevari, S.;Fossati, G.; Colnaghi, M. I. Cancer Res. 1983, 43, 1295. (d) Zhang, S.;Zhang, H. S.; Cordon-Cardo, C.; Ragupathi, G.; Livingston, P. O. Clin.Cancer Res. 1998, 4, 2669.) H1299 cells, a model for non-small cell lungcarcinoma and also reported to express Globo H, (Lee J. S., R. J. Y.,Sahin A. A., Hong, W. K., Brown, B. W., Mountain, C. F., Hittleman, W.N. N. Engl. J. Med. 1991, 324, 1084.) showed lower Fucα(1-2)Galexpression. Importantly, flow cytometry analysis revealed a 53% increasein Fucα(1-2)Gal expression on the surface of LnCAP cells compared tonon-cancerous PrEC cells. These results demonstrate that ourchemoenzymatic labeling approach can readily discriminate cancerouscells from normal cells, providing a new potential strategy for rapidbiomarker detection. The method could be particularly useful for thedetection of prostate cancer from tissue biopsies, as the currentstandard of PSA detection to diagnose prostate cancer has a significantfalse-positive rate, leading to overtreatment. (Schröder, F. H. et al.N. Engl. J. Med. 2009, 360, 1320.) In addition to histologicaldetection, our chemoenzymatic approach could potentially provide a newstrategy to distinguish normal PSA from tumorigenic PSA, which isreported to have higher levels of Fucα(1-2)Gal glycosylation.(Peracaula, R.; Tabares, G.; Royle, L.; Harvey, D. J.; Dwek, R. A.;Rudd, P. M.; de Llorens, R. Glycobiology 2003, 13, 457.)

In conclusion, we have developed a new chemoenzymatic strategy thatdetects Fucα(1-2)Gal glycans with improved efficiency and selectivityover existing methods. Our strategy detects a variety of complexFucα(1-2)Gal glycans and glycoproteins and permits living cells orcomplex tissue extracts to be rapidly interrogated. We anticipate thatthe strategy will accelerate both the discovery of new Fucα(1-2)Galglycoproteins and advance an understanding of the biological roles ofthis important sugar in neurobiology and cancer. Moreover, this studyrepresents a proof-of-concept that chemoenzymatic labeling strategiescan be extended to more complex oligosaccharides. Future studies willexpand chemoenzymatic detection approaches to a broad range of glycansto provide a powerful new set of tools for glycomics research.

General Methods for Chemical Synthesis.

Unless otherwise stated, all starting materials and reagents werepurchased from Sigma-Aldrich and used without further purification. All¹H and ¹³C NMR spectra were recorded on a Varian Innova 600 spectrometerand referenced to solvent peaks. Data for ¹H NMR spectra are reported asfollows: chemical shift (6 ppm), multiplicity (s=singlet, d=doublet,t=triplet, q=quartet, m=multiplet), coupling constant in Hz, andintegration. Low-resolution mass spectra were recorded on an 1100Agilent Liquid Chromatograph Mass Spectrometer with an Agilent SB-C18reverse-phase column (3.5 μm, 4.6×250 mm) with monitoring at 280 and 310nm. High-resolution mass spectra (HRMS) were obtained using an Agilent6200 Series Time of Flight Mass Spectrometer with an Agilent G1978AMultimode source using mixed electrospray ionization/atmosphericpressure chemical ionization (MultiMode ESI/APCI).

Synthesis of 1-p-Nitrobenzyl-(2-fucosyl)-lactose (1)

A 0.35 M solution of p-nitrobenzylamine in 7:3 (v/v) DMSO/AcOH (50 μL 18μmol) was added slowly to 2′-fucosyllactose (1.0 mg, 2.0 μmol) at rt.NaCNBH₃ (50 μL of a 1 M solution in 7:3 (v/v) DMSO/AcOH, 50 μmol) wasthen added slowly at rt, and the solution was stirred at 65° C. for 4 h.The reaction was quenched by adding 10 volumes of MeCN and incubated at−20° C. for 2 h. The precipitated mixture was then centrifuged at10,000×g for 5 min at 4° C., and the supernatant was discarded. Tenadditional volumes of MeCN were added to the pellet, and the vortexedmixture was incubated at −20° C. for 2 h and centrifuged as above. Thisstep was repeated two more times to remove the excessp-nitrobenzylamine. The pellet was then resuspended in 5% MeCN and theproduct purified by semi-preparative HPLC (Agilent 1100) using twopreparative reverse-phase columns (Agilent Eclipse XDB-C18; 5 μm,9.4×250 mm) connected in series and a gradient of 5-20% B over 20 min at4 mL/min (A, 0.5% aqueous AcOH; B, 100% MeCN). The product eluted atapproximately 9.5 min. Lyophilization afforded a fluffy white solid(0.72 mg; 56% yield): ¹H NMR (600 MHz, D₂O) δ 8.33 (d, J=8.6 Hz, 2H),7.72 (d, J=8.7 Hz, 2H), 5.31 (s, 1H), 4.60 (d, J=7.8 Hz, 1H), 4.33 (q,J=13.7 Hz, 2H), 4.23 (t, J=6.3 Hz, 2H), 3.93 (d, J=3.4 Hz, 1H),3.91-3.86 (m, 4H), 3.83 (t, J=4.4 Hz, 4H), 3.79-3.71 (m, 4H), 3.67 (dd,J=9.5, 7.9 Hz, 1H), 3.32 (d, J=11.7 Hz, 1H), 3.10 (t, J=11.1 Hz, 1H),1.23 (d, J=6.6 Hz, 3H). ¹³C NMR (151 MHz, D₂O) δ 147.86, 130.43, 124.09,109.99, 107.14, 100.42, 99.65, 76.94, 76.65, 75.19, 73.42, 71.69, 70.48,70.19, 69.45, 68.97, 68.51, 68.21, 67.18, 61.93, 60.90, 50.51, 49.52,23.19, 15.29. HRMS: [M+H]⁺ calculated for C₂₅H₄₀N₂O₁₆ 625.5996. Found625.2451.

Expression and Purification of BgtA.

E. coli BL21 (DE3) harboring the recombinant plasmid vectorpET28a-BtgA-His was kindly provided by Dr. Peng George Wang (Ohio StateUniversity). The protein was expressed and purified as described. (Yi,W.; Shen, J.; Zhou, G.; Li, J.; Wang, P. G. J. Am. Chem. Soc. 2008, 130,14420.) Briefly, the cells were grown in LB medium (1 L) at 37° C.Isopropyl-1-thio-β-D-galactospyranoside (IPTG, 0.8 mM finalconcentration; Sigma) was added when the cells reached an OD₆₀₀ of 0.8,and the cells were incubated for an additional 18 h at 16° C. Thepelleted cells were lysed in Cell Lytic B Lysis Reagent (Sigma-Aldrich)supplemented with EDTA-free Complete™ protease inhibitors (Roche), 0.5 MNaCl, and 20 mM imidazole (Sigma-Aldrich) by rotating end-over-end for20 min at rt. After centrifugation, the clarified lysate was added toprewashed Ni-NTA beads and incubated at 4° C. for 1 h, washed in 20 mMTris.HCl pH 7.5, 0.5 M NaCl and 50 mM imidazole, and eluted in a stepgradient with the elution buffer (20 mM Tris.HCl pH 7.5, 0.5 M NaCl, and100, 200 or 500 mM imidazole). After SDS-PAGE analysis, the purifiedprotein was concentrated with 10,000 Da molecular weight cut-off (MWCO)spin filters (Millipore) and dialyzed into 20 mM Tris.HCl pH 7.5containing 10% glycerol and stored at 4° C.

BgtA Activity Assay and Monitoring of Chemoenzymatic Labeling Reactionsby LC-MS/MS.

The Fucα(1-2)Gal substrate 1 (10 μM) was dissolved in 20 mM Tris.HCl pH7.5, 50 mM NaCl, and 5 mM MnCl₂. BgtA enzyme (Yi, W.; Shen, J.; Zhou,G.; Li, J.; Wang, P. G. J. Am. Chem. Soc. 2008, 130, 14420.) andUDP-ketoGal 3 (Khidekel, N.; Arndt, S.; Lamarre-Vincent, N.; Lippert,A.; Poulin-Kerstien, K. G.; Ramakrishnan, B.; Qasba, P. K.;Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2003, 125, 16162.) or UDP-GalNAz 2(Invitrogen) were added to final concentrations of 0.16 mg/mL and 50 μM,respectively, in a final volume of 100 μL. The reaction was incubated at4° C. in the dark for 12-16 h, and the reaction progress was monitoredby LC-MS/MS. To label with aminooxy-biotin 7, the reactions were diluted5-fold with saturated urea, 2.7 M NaOAc pH 3.9 (50 mM finalconcentration and pH 4.8), and 7 (5 mM final concentration, Dojindo) andincubated for 20-24 h at rt. To label with ADIBO-biotin 6, 250 μM of 6(Click Chemistry Tools) was added, and the reaction was incubated for 3h at rt. Following the labeling steps, the azido-labeled samples werefiltered through a 3,000 Da MWCO Vivaspin 500 spin filter (GELifesciences) and injected on a reverse-phase HPLC column (PhenomenexGemini; 5 m, 2.0×100 mm), fitted with a C8 guard column, using aThermoScientific Accela 600 HPLC pump interfaced with a ThermoScientificLTQ mass spectrometer. A linear 3-90% gradient of B (A: 0.1% aqueousformic acid, B: 0.1% formic acid in MeCN) over 7 min was used to resolvepeaks with a flow rate of 0.21 mL/min. Mass analysis was performed inpositive ion mode except in the case of sulfated compound 8, where theanalysis was performed in negative ion mode.

Kinetic Analysis of BgtA with UDP-GalNAz and UDP-GalNAc.

Reactions were performed in duplicate with 100 μM acceptor substrate(1), 0.7 μg BgtA, and varying concentrations of UDP-GalNAz or UDP-GalNAc(50 to 800 μM) in 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MnCl₂ at rtin a total volume of 20 μL. Product formation was monitored at 280 nm byreverse phase-HPLC (Agilent 1100), and time points were taken over thecourse of 5 min using a linear, 3-95% gradient of B (A: 0.1% aqueoustrifluoroacetic acid B: 0.1% trifluoroacetic acid in MeCN) over 8 minwith a flow rate of 1 mL/min. The kinetic parameters, apparent K_(m),V_(max), and k_(cat), were obtained by linear regression analysis ofinitial velocity vs. donor substrate concentration using KaleidaGraph(version 4.1.2).

Chemoenzymatic Labeling on the Glycan Array.

Glycan Array version 5.0 was provided by the Consortium for FunctionalGlycomics (CFG). Pre-equilibrated arrays were treated with BgtA enzyme(0.16 mg/mL) and 500 μM UDP-GalNAz 2 in 20 mM Tris.HCl pH 7.4, 50 mMNaCl, 2 mM MnCl₂ containing 1% bovine serum albumin (BSA) for varioustimes (0, 0.5, 2, and 12 h) at rt, washed 4 times with wash buffer (20mM Tris.HCl pH 7.4, 50 mM NaCl, 0.1% Triton X-100) and then 4 times withrinse buffer (20 mM Tris.HCl pH 7.4, 50 mM NaCl). The arrays were thenincubated with ADIBO-biotin (5 μM) in 20 mM Tris.HCl pH 7.4, 50 mM NaClfor 2 h at rt. After washing as described above, the arrays were washedfurther with 20% aqueous MeOH and then incubated with streptavidin Cy-5(0.5 μg/mL; eBioSciences) in 20 mM Tris.HCl pH 7.4, 50 mM NaCl, 0.05%Tween-20, 1% BSA, for 1 h at rt. The arrays were then washed 4 timeswith wash buffer (containing 0.05% Tween-20 instead of 0.1% TritonX-100), 4 times with rinse buffer, 4 times with water, dried under a lowstream of filtered air, and scanned using a PerkinElmer ScanArrayExpress fluorescence scanner and ImaGene data analysis software(BioDiscovery). Data were analyzed per the guidelines of the CFG. ((a)Blixt, O.; Head, S.; Mondala, T.; Scanlan, C.; Huflejt, M. E.; Alvarez,R.; Bryan, M. C.; Fazio, F.; Calarese, D.; Stevens, J.; Razi, N.;Stevens, D. J.; Skehel, J. J.; van Die, I.; Burton, D. R.; Wilson, I.A.; Cummings, R.; Bovin, N.; Wong, C.-H.; Paulson, J. C. Proc. NatlAcad. Sci. USA 2004, 101, 17033. (b) Smith, D. S.; Song, X.; Cummings,R. D. Methods in Enzymology 2010, 480, 417.) The background fluorescencefor each spot was determined as the fluorescence signal outside of thearea designated as a positive spot on the array. Because backgroundfluorescence can be heterogeneous across the array, each array wasdivided into areas of 10 by 10 spots, or sub-arrays, and a backgroundfluorescence value was calculated within each sub-array by the software.This value was then subtracted from the fluorescence signal for eachglycan spot within the sub-array by the software. Note that each glycanis printed six different times on the array. To calculate the relativefluorescence intensity for a given glycan, the highest and lowestintensities for each glycan were discarded, and the mean and standarddeviation of four fluorescence intensities were calculated.

Chemoenzymatic Labeling of Cell Lysates.

The olfactory bulbs of postnatal day 3 rat pups were dissected on iceand lysed in boiling 1% SDS (5 volumes/weight) with sonication until themixture was homogeneous. Protein was precipitated usingmethanol/chloroform/water. Briefly, protein was diluted to 200 μL andprecipitated by sequential mixing with 600 μL of MeOH, 200 μL of CHCl₃and 450 μL H₂O, after which the mixture was centrifuged at 23,000×g for15 min. Precipitated protein was washed with 450 μL of MeOH andcentrifuged at 23,000×g for 10 min. After the protein pellet was allowedto dry briefly, the pellet was re-dissolved at 5 mg/mL in 20 mM HEPES pH7.9 containing 1% SDS, and diluted 5-fold into a buffer with thefollowing final concentrations: 20 mM HEPES pH 7.9, 50 mM NaCl, 2%NP-40, 5 mM MnCl₂. UDP-GalNAz 2 (25 μM; Invitrogen) and BgtA (0.16mg/mL) were added, and the samples were incubated at 4° C. for 16-20 h.The labeled proteins were precipitated as above and resuspended in 50 mMTris pH 7.4 containing 1% SDS at 4 mg/mL.

The resuspended proteins were subsequently reacted with alkyne-TAMRA 10(Invitrogen) or alkyne-biotin 11 (Invitrogen) as per the Click-It™ TAMRAand Biotin Glycoprotein Detection Kit (Invitrogen) instructions, exceptthat EDTA-free Complete™ protease inhibitors were added during thereaction. For TAMRA labeling, negative controls were performed underidentical conditions except that BgtA, UDP-GalNAz 2, or alkyne-TAMRA 10was omitted from the labeling reaction. After the labeling reactions,protein was precipitated using chloroform/methanol/water as describedabove and re-dissolved in boiling 2% SDS. This precipitation andresolubilization was then repeated once more to ensure removal ofnon-specific interactions. TAMRA-labeled proteins were resolved bySDS-PAGE and visualized in-gel using a Typhoon Scanner (GE Healthcare).Interestingly, BgtA itself was labeled, and attempts to remove thesignal by precipitation or heat and detergent denaturation wereunsuccessful, suggesting covalent modification of the protein. Forbiotin labeling, negative controls were performed under identicalconditions except that BgtA was omitted from the labeling reaction.

Purification of Biotin-Labeled Fucα(1-2)Gal Proteins.

Chemoenzymatically labeled samples were precipitated usingmethanol/chloroform/water as described above and re-dissolved in boiling1% SDS plus Complete™ protease inhibitors at a concentration of 2 mg/mL.The SDS was quenched with 1 volume of NETFD buffer (100 mM NaCl, 50 mMTris.HCl pH 7.4, 5 mM EDTA, 6% NP-40) plus protease inhibitors. Thesamples were incubated with pre-washed streptavidin resin (Pierce; 100μL/1 mg protein) for 2 h at 4° C. The resin was washed twice with 10column volumes each of low salt buffer (0.1 M Na₂HPO₄ pH 7.5, 0.15 MNaCl, 1% Triton-X100, 0.1% SDS), twice with 10 column volumes each ofhigh salt buffer (0.1 M Na₂HPO₄ pH 7.5, 0.5 M NaCl, 0.2% Triton-X100),and once with 10 column volumes of 50 mM Tris.HCl pH 7.4. Capturedprotein was eluted in boiling 2× sample buffer (100 mM Tris pH 6.8, 4%SDS, 200 mM DTT, 20% glycerol, 0.1% bromophenol blue; 50 μL/100 μLresin) for 5 min.

Western Blotting for Parallel Identification of Fucα(1-2)GalGlycoproteins.

The purified, labeled material from above was resolved on a NuPAGE 4-12%Bis-Tris gel (Invitrogen) and transferred to a polyvinylidene difluoride(PVDF) membrane (Millipore). The membrane was blocked in 5% milk(BioRad) in TBST (50 mM Tris.HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.4)for 1 h at rt. Primary antibodies in 5% milk in TBST were addedovernight at 4° C. at the following concentrations: mouse anti-NCAMmonoclonal antibody (Abcam) at 1 μg/mL, mouse anti-synapsin I ascites(Synaptic Systems) at 0.1 μg/ml, mouse anti-munc18-1 (Synaptic Systems)at 0.1 μg/mL, or mouse p44 MAPK monoclonal antibody (Cell Signaling) at1:2000 dilution. Membranes were washed with TBST, and incubated with theappropriate Alexa Fluor 680-conjugated (Invitrogen) or IR800-conjugated(Rockland) secondary antibody, and visualized using a LiCOR OdysseyImaging System.

Lectin Affinity Chromatography with UEAI.

UEAI lectin conjugated to agarose (Vector Laboratories) or controlprotein A conjugated to agarose (Vector Laboratories) was packed into aminicolumn (50 μL bed volume; Bio-Rad), and the columns were run inparallel. The resin was equilibrated with 10 column volumes of lectinbinding buffer (100 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂,0.5% NP-40, and 0.2% sodium deoxycholate supplemented with EDTA-freeComplete™ protease inhibitors). Olfactory bulb tissue from P3 rat pupswas lysed in lectin binding buffer by sonication on ice. The lysate wasclarified by centrifugation at 12000×g for 10 min, and the total proteinconcentration was determined using the BCA protein assay (Pierce).Lysate (500 μg) was bound batch-wise with gentle end-over-end mixing atrt for 4 h. The agarose was then allowed to settle, and the flow-throughwas passed over the column three additional times. The columns werewashed with 40 column volumes of lectin binding buffer, followed by 10column volumes of lectin binding buffer lacking detergent. Proteins wereeluted in 10 column volumes of lectin binding buffer lacking detergentand supplemented with 200 mM L-fucose and Complete™ protease inhibitors.Protein eluates were concentrated to a volume of 100 μL using a Vivaspin500 spin filter (10,000 Da MWCO). Following concentration, samples wereboiled in 1× sample buffer (35 μL of 200 mM Tris pH 6.8, 400 mM DTT, 8%SDS, 0.2% bromophenol blue, and 40% glycerol) and analyzed by SDS-PAGEand western blotting as described above. Synapsin I was detected usingmouse anti-synapsin I ascites (Synaptic Systems) at 0.1 μg/mL.

Cell Culture.

HeLa, MCF-7, and MDA-mb-231 cells grown in DMEM medium supplemented with10% fetal bovine serum (FBS), 100 units/mL penicillin, and 0.1 mg/mLstreptomycin (Gibco). LNCaP and H1299 cells were grown in RPMI medium1640 supplemented with 10% FBS, 100 units/mL penicillin, and 0.1 mg/mLstreptomycin (Gibco). The PrEC line was maintained in PrEBM medium(Lonza). All transfections were carried out in antibiotic-free media. Inall cases, cells were incubated in a 5% CO₂ humidified chamber at 37° C.The PrEC line was obtained from Lonza; all other cell lines wereobtained from ATCC.

Immunoprecipitation of TAMRA-Labeled Synapsin I from HeLa Cell Lysates.

HeLa cells were transfected with pCMV-FLAG-synapsin Ia (Murrey, H. E.;Gama, C. I.; Kalovidouris, S. A.; Luo, W.-I.; Driggers, E. M.; Porton,B.; Hsieh-Wilson, L. C. Proc. Natl Acad. Sci. USA 2006, 103, 21.) usingLipofectamine LTX reagent (Invitrogen). The cells were lysed andchemoenzymatically labeled and protein was precipitated as describedabove. After the protein pellet was allowed to dry briefly, the pelletwas re-dissolved in boiling 1% SDS plus Complete™ protease inhibitors ata concentration of 2 mg/mL. The SDS was quenched with 1 volume of NETFDbuffer plus protease inhibitors, and the lysate was incubated with 40 μLof prewashed anti-Flag M2 Affinity Gel (Sigma-Aldrich) for 90 min at 4°C. The resin was washed once with 4 column volumes of NETFD buffer andthree times with 4 column volumes of NETF buffer (100 mM NaCl, 50 mMTris.HCl pH 7.4, 5 mM EDTA). Captured protein was eluted in boiling 2×sample buffer (50 μL buffer/100 μL resin). Purified, labeled materialwas resolved by SDS-PAGE and transferred to a polyvinylidene fluoride(PVDF) membrane (Millipore). Western blotting was performed as aboveexcept the primary anti-TAMRA rabbit antibody (0.1 μg/μL; Invitrogen)was used.

Detection of Cell-Surface Fucα(1-2)Gal Glycans on Live MCF-7 Cells byFluorescence Microscopy.

MCF-7 cells (ATCC) were seeded at 2×10⁵ cells/coverslip. Twelve hoursafter plating, the cells were washed twice with 1% FBS, 10 mM HEPES incalcium and magnesium free Hank's Balanced Salt Solution (CMF HBSS,Gibco) and incubated in the chemoenzymatic labeling buffer (2% FBS, 10mM HEPES pH 7.9 in HBSS) with UDP-GalNAz 2 (500 μM) and BgtA (0.17mg/mL) in a total volume of 100 μL for 2 h at 37° C. Mock reactions wereperformed without the addition of BgtA. After chemoenzymatic labeling,the cells were washed twice with 100% FBS and twice with thechemoenzymatic labeling buffer. Enzymatic addition of GalNAz ontoFucα(1-2)Gal glycans was detected by incubating the cells withADIBO-biotin (20 μM in the chemoenzymatic labeling buffer; 500 μL) for 1h at rt, washing the coverslips as described, and then incubation withstreptavidin-Alexa Fluor 488 (1 μg/mL in PBS containing 3% BSA;Invitrogen) for 30 min at rt. Cells were washed once with PBS, afterwhich nuclei were stained with Hoechst-33342 (1 μg/μL; Invitrogen) inPBS for 15 min at rt. Coverslips were washed twice with 100% FBS andmounted in media (on ice), sealed with paraffin, and imaged immediatelyusing a 40× Plan-Achromat objective on a Zeiss Meta510 invertedmicroscope.

Detection of Fucα(1-2)-Gal Glycans on Synapsin I in Fixed HeLa Cells byFluorescence Microscopy.

HeLa cells were plated onto 15 mm coverslips (Carolina Biologicals) at adensity of 75 cells/mm². After 12 h, cells were transfected withpCMV-Flag-synapsin Ia (0.5 μg DNA/coverslip) using Lipofectamine LTX (4μL in 200 μL Optimem; Invitrogen). After 24 h, the media was removed,and the cells were rinsed one time with PBS, fixed in 4%paraformaldehyde in PBS, pH 7.5 for 20 min at rt, washed twice with PBS,permeabilized in 0.3% Triton X-100 in PBS for 10 min at rt, and washedtwice with the enzymatic labeling buffer (50 mM HEPES, 125 mM NaCl, pH7.9). Reaction mixtures and negative controls (without BgtA) wereprepared by adding 100 μL of 20 mM HEPES pH 7.9, 50 mM NaCl, 2% NP-40, 5mM MnCl₂, UDP-GalNAz 1 (25 μM), and BgtA (0.17 mg/mL) at 4° C. for 24 h(100 μL/coverslip) in a humidified chamber. After chemoenzymaticlabeling, the cells were washed twice with the chemoenzymatic labelingbuffer. Enzymatic addition of GalNAz onto Fucα(1-2)Gal glycans wasdetected by treating the cells with 5 μM alkyne-functionalized AlexaFluor 488 (Invitrogen), 0.1 mM triazoleamine ligand (Invitrogen), 2 mMsodium ascorbate (Sigma-Aldrich), and 1 mM CuSO₄ (Sigma-Aldrich) in 2%FBS (Gibco) in PBS at rt for 1 h. Synapsin I was detected byimmunostaining with an anti-synapsin I antibody (Millipore, 1:250 in 3%BSA) for 1 h at rt, followed by an anti-rabbit secondary antibodyconjugated to Alexa Fluor 546 (Invitrogen; 1:1000 in 3% BSA) for 1 h atrt. The coverslips were washed with PBS, mounted onto glass slides usingVectashield mounting medium with DAPI (4 μL; Vector Labs) and sealedwith clear nail polish. Cells were imaged using a Nikon Eclipse TE2000-Sinverted microscope, and images were captured with Metamorph softwareusing a 20× Plan Fluor objective.

Detection of Cell-Surface Fucα(1-2)Gal Glycans on Live Cancer Cells byFlow Cytometry.

All cells were seeded at 4×10⁶ cells per 10-cm plate in 10 mL of theappropriate media. On the day of analysis, cells were lifted off theplate with DNase (0.4 mg/mL; Worthington) and 1 mM EDTA and washed with1% FBS, 10 mM HEPES in CMF HBSS. One million cells werechemoenzymatically labeled with UDP-GalNAz (500 μM) and BgtA (0.17μg/μL) in 1% FBS, 10 mM HEPES in CMF HBSS (100 μL) for 2 h at 37° C.Cells were spun twice through 100% FBS (1 mL) to remove excess reagent(500×g, 5 min) and resuspended in 1% FBS, 10 mM HEPES in CMF HBSS (100μL) containing ADIBO-biotin (20 μM) and incubated for 1 h at rt. Cellswere again spun twice through 100% FBS (1 mL), and washed with 3% BSA inPBS (1 mL). Cells were then resuspended in 3% BSA in PBS (100 μL)containing streptavidin-Alexa Fluor 488 (1 μg/mL) and incubated for 20min at 4° C. Cells were subsequently spun twice through 100% FBS (1 mL)and resuspended in 2% FBS, 10 mM HEPES in CMF HBSS (750 μL) for flowcytometry analysis. Immediately before analysis, 7-amino-actinomycin D(7-AAD, 5 μL; eBioscience) was added to measure cell viability. Cellswere analyzed for FITC intensity on a Beckman Dickenson FACSCalibur flowcytometer equipped with a 488-nm argon laser. For each experiment,10,000 live cells were analyzed, and data analysis was performed onFlowJo (Tristar Inc.). Data points for LnCAP and PrEC cells werecollected in triplicate, and for all other cells, in duplicate.

We have developed a chemoenzymatic approach for the detection ofglyco-conjugates containing the fucose-α(1-2)-galactose (Fucα(1,2)Gal)motif. Using the bacterial homologue to the blood group transferase A(BgtA), we can specifically install azido- or ketone-containing sugars(e.g., GalNAz, or 2-deoxy-keto-Gal) onto the disaccharide moiety.Following the use of “click chemistry” (Cu(I)-catalyzed orstrain-promoted [3+2] cycloaddition chemistry) or oxime chemistry withdetection reagents (affinity, fluorescent, mass tag, isotope tag, etc.)we can detect this glycan structure using various readouts (e.g.,Western Blotting, in gel fluorescence, flow cytometry, massspectrometry, and fluorescence microscopy).

We envision many other applications of our method. First, this method isamenable to proteomics. Following the labeling of Fucα(1,2)Gal the withaffinity reagents such as biotin or TAMRA, proteins may be purified,digested with a protease, and the Fucα(1,2)Gal proteins identified bymass spectrometry. Alternatively, labeled proteins can be digested withproteases and the glycopeptides enriched by affinity chromatography(e.g., avidin agarose or using an anti-TAMRA antibody) and subjected tomass spectrometry to identify the corresponding Fucα(1,2)Gal proteins.Following this experimental workflow this method can also be used foridentifying the site of glycosylation on the protein, that is, whatamino acid side chain is modified with this glycan, as well as theentire Fucα(1,2)Gal-containing glycan. These proteomic studies would beinterested for identifying glycoproteins from serum or tumors that haveincreasing amounts of Fucα(1,2)Gal epitope in various cancers, stages ofcancer, or other disease states.

This method can also be used to identify changes in the levels ofFucα(1,2)Gal glycoconjugates on tissues or cells after variousstimulations, such as neuronal activity or learning, cancer treatments(a method to identify efficacy of cancer treatment in patients), andpharmacological treatments (studying what signaling pathways in cellslead to differential expression of these glycans on the cell surface).This can be done using methods known in the field such as quantitativeproteomics, Western blotting and/or in gel fluorescence.

Our method can also allow quantification of the stoichiometry ofFucα(1,2)Gal on specific proteins following the outline described inRexach et al. (Nature Chemical Biology 2010, 9, 645-651). This methodemploys installing a polyethyleneglycol polymer mass tag instead of anaffinity or fluorescent tag, immunoblotting for protein(s) of interest,and quantifying the relative intensities of the mass shifted bands toquantify the stoichiometry of glycosylation.

This labeling technology may also be applied as a diagnostic tool todetect the presence of Fucα(1,2)Gal on potential biomarkers. Forexample, fluorescently labeled Fucα(1,2)Gal proteins from cell lysatesor serum could be captured (e.g., using antibodies against the proteinof interest, such as against prostate specific antigen, PSA, forprostate cancer) on the bottom of a 96-well plate or other format, andthe levels of Fucα(1,2)Gal on the protein of interest could be detectedby fluorescence.

In summary the potential applications of this labeling method are assuch:

1) Proteomics/Lipidomics—identification of glycoproteins/biomarkersbearing Fucα(1,2)Gal.

2) Site mapping—identification of amino acids modified by fucα(1,2)gal.

3) Monitoring changes in Fucα(1,2)Gal levels following variousstimulations or normal versus disease states.

4) Characterizing the stoichiometry of the Fucα(1,2)Gal modification onspecific proteins.

5) Use of this method as a diagnostic tool for detecting biomarkers inan ELISA assay-type format. Alterations in Fucα(1,2)Gal glycanexpression could occur in the case of various cancers, learning, aging,neurodegenerative diseases and other diseases.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

SEQUENCE LISTING Helicobacter mustelae homologue of Human blood Group A transferase (BgtA) SEQ ID NO: 1MQSTAQNTQQNTHFAGSSQTTPQAAQSVQQASLALPKSSPTCYKIAILYICTGAYSIFWQDFYDSAKVHLLPAHRLTYFVFTDADSLYAEEASDVRKIYQENLGWPFNTLKRFEMFLGQEEALREFDFVFFFNANCLFFQHIGDEFLPIEEDILVTQHYGFRDASPECFTYERNPKSLAYVPFGKGKAYVYGSTNGGKAGAFLALARTLQERIQEDLSRGIIAIWHDESHLNAYIIDHPNYKMLDYGYGFPEGYGRVPGGGVYIFLRDKSRVIDVNAIKGMGSPANRRLKNALRKLKHFSKRLLGRHelicobacter mustelae homologue of Human blood Group A transferase (BgtA)(Pet28a-BgtA-6His (“6His”disclosed as SEQ ID NO: 6))(this isthe enzyme we actually use after cloning into Pet28a vector SEQ ID NO: 2MGQSTAQNTQQNTHFAGSSQTTPQAAQSVQQASLALPKSSPTCYKIAILYICTGAYSIFWQDFYDSAKVHLLPAHRLTYFVFTDADSLYAEEASDVRKIYQENLGWPFNTLKRFEMFLGQEEALREFDFVFFFNANCLFFQHIGDEFLPIEEDILVTQHYGFRDASPECFTYERNPKSLAYVPFGKGKAYVYGSTNGGKAGAFLALARTLQERIQEDLSRGIIAIWHDESHLNAYIIDHPNYKMLDYGYGFPEGYGRVPGGGVYIFLRDKSRVIDVNAIKGMGSPANRRLKNALRKLKHFSKRLLGRLEHHHHHH Human blood Group A transferaseSEQ ID NO: 3 MAEVLRTLAGKPKCHALRPMILFLIMLVLVLFGYGVLSPRSLMPGSLERGFCMAVREPDHLQRVSLPRMVYPQPKVLTPCRKDVLVVTPWLAPIVWEGTFNIDILNEQFRLQNTTIGLTVFAIKKYVAFLKLFLETAEKHFMVGHRVHYYVFTDQPAAVPRVTLGTGRQLSVLEVRAYKRWQDVSMRRMEMISDFCERRFLSEVDYLVCVDVDMEFRDHVGVEILTPLFGTLHPGFYGSSREAFTYERRPQSQAYIPKDEGDFYYLGGFFGGSVQEVQRLTRACHQAMMVDQANGIEAVWHDESHLNKYLLRHKPTKVLSPEYLWDQQLLGWPAVLRKLRFTAVPKNHQAVRNP Rat blood Group A transferase SEQ ID NO: 4MDLRGRPKCYSLHLGILPFIVLVLVFFGYGFLSHKIQEFRNPGGETCMATRQTDVQKVVSVPRMAYPQPNVLTPIRNDVLVFTPWLAPIIWEGTFNIDILNEQFKLQNTTIGLTVFAIKKYVVFLKLFLETAEQHFMVGHKVIYYVFTDRPSDVPQVPLGAGRKLVVLTVRNYTRWQDVSMHRMEMISHFSEQRFQHEVDYLVCGDVDMKFSDHVGVEILSALFGTLHPGFYRSRRESFTYERRPKSQAYIPRDEGDFYYAGGFFGGSVVEVHHLTKACHQAMVEDQANGIEAVWHDESHLNKYLLYHKPTKVLSPEYVWDQKLLGWPSIMKKLRYVAVPKNHQAIRN Mouse blood Group A transferase SEQ ID NO: 5MNLRGRPKCNFLHLGILPFAVFVLVFFGYLFLSFRSQNLGHPGAVTRNAYLQPRVLKPTRKDVLVLTPWLAPIIWEGTFNIDILNEQFRIRNTTIGLTVFAIKKYVVFLKLFLETAEQHFMVGHKVIYYVFTDRPADVPQVILGAGRQLVVLTVRNYTRWQDVSMHRMEMISHFSERRFLREVDYLVCADADMKFSDHVGVEILSTFFGTLHPGFYSSSREAFTYERRPQSQAYIPWDRGDFYYGGAFFGGSVLEVYHLTKACHEAMMEDKANGIEPVWHDESYLNKYLLYHKPTKVLSPEYLWDQQL LGWPSIMKKLRYVAVPKDHQAIRN

What is claimed is:
 1. A glycan comprising: 1) a first glycosyl groupcomprising fucose-α(1-2)-galactose; and 2) a second glycosyl groupcovalently linked to the first glycosyl group, wherein the secondglycosyl group is covalently linked to the first glycosyl group at theC-3 position of the galactose on the first glycosyl group, and whereinthe second glycosyl group comprises a reactive group (R), whereinlinking the second glycosyl group to the first glycosyl group involvesreacting a labeling agent with a glycosyltransferase, wherein thelabeling agent is a compound of the formula:

wherein R is a substituent selected from the group consisting of astraight chain or branched C₁-C₁₂ carbon chain bearing a carbonyl group,an azide group, a straight chain or branched C₁-C₁₂ carbon chain bearingan azide group, a straight chain or branched C₁-C₁₂ carbon chain bearingan alkyne, or a straight chain or branched C₁-C₁₂ carbon chain bearingan alkene.
 2. The glycan of claim 1, wherein the glycan is attached to aglycoprotein or glycolipid.
 3. The glycan of claim 1, further comprisinga detection agent covalently linked to the second glycosyl group,wherein the detection agent is covalently linked to the second glycosylgroup via a reaction between a coupling moiety on the detection agentand the reactive group, and wherein the detection agent is selected fromthe group consisting of a fluorescent reagent, an enzymatic reagentcapable of converting substrates calorimetrically or fluorometrically, afluorescent and luminescent probe, a metal-binding probe, aprotein-binding probe, a probe for antibody-based binding, a radioactiveprobe, a photocaged probe, a spin-label, spectroscopic probe, aheavy-atom containing probe, poly(ethylene)glycol-containing probe andpoly(propylene)glycol-containing probe.
 4. The glycan of claim 1,wherein the glycan has the formula of


5. The glycan of claim 1, wherein the glycosyltransferase is specificfor the fucose-α(1-2)-galactose group.
 6. The glycan of claim 1, whereinthe glycosyl transferase is a bacteria homologue of the human bloodgroup A antigen glycosyltransferase (BgtA) or a variant or fragmentthereof.
 7. The glycan of claim 1, wherein the glycosyltransferase is ahuman blood group A antigen glycosyltransferase or a variant or fragmentthereof.
 8. A reaction mixture for generating the glycan of claim 4comprising 1) fucose-α(1-2)-galactose; 2) a labeling agent of theformula:

wherein R is a reactive group, wherein R is a substituent selected fromthe group consisting of a straight chain or branched C₁-C₁₂ carbon chainbearing a carbonyl group, an azide group, a straight chain or branchedC₁-C₁₂ carbon chain bearing an azide group, a straight chain or branchedC₁-C₁₂ carbon chain bearing an alkyne, or a straight chain or branchedC₁-C₁₂ carbon chain bearing an alkene; wherein the labeling agent isrecognized by the glycosyltransferase.
 9. The mixture of claim 8,wherein the glycan is attached to a glycoprotein or glycolipid.
 10. Thereaction mixture of claim 8, further comprising a detection agent, andwherein the detection agent is selected from the group consisting of afluorescent reagent, enzymatic reagent capable of converting substratescalorimetrically or fluorometrically, fluorescent and luminescent probe,metal-binding probe, protein-binding probe, probe for antibody-basedbinding, radioactive probe, photocaged probe, spin-label, spectroscopicprobe, a heavy-atom containing probe, poly(ethylene)glycol-containingprobe and poly(propylene)glycol-containing probe.
 11. The reactionmixture of claim 8, wherein labeling agent is placed on a solid support.12. A method for labeling a glycan comprising fucose-α(1-2)-galactose,the method comprising: reacting the fucose-α(1-2)-galactose with alabeling agent in the presence of a glycosyltransferase, wherein thelabeling agent is a compound of the formula:

wherein R is a reactive group selected from the group consisting of astraight chain or branched C₁-C₁₂ carbon chain bearing a carbonyl group,an azide group, a straight chain or branched C₁-C₁₂ carbon chain bearingan azide group, a straight chain or branched C₁-C₁₂ carbon chain bearingan alkyne, or a straight chain or branched C₁-C₁₂ carbon chain bearingan alkene; wherein the labeling agent is recognized by theglycosyltransferase; wherein the reactive group is capable of reactingwith a detection agent; and wherein the glycosyltransferase is specificfor the glycosyl group labeling agent.